Design and evaluation of primer pairs for efficient detection of avian rotavirus

The use of molecular methods for rotavirus characterisation provides increased sensitivity for typing and allows the identification of putative reassortant strains. Reagents and methods for genotyping the virus need constant modification because of the reassortant nature of the virus. This study was aimed at designing and evaluating new oligonucleotide degenerate primer pairs that provide increased sensitivity and specificity for detecting avian rotavirus. Gene-specific primer pairs were designed by analysing different rotavirus strains isolated during the last decade by downloading them from the GenBank. The alignments were generated using clustal analysis from the BioEdit program. Degenerate nucleotides were included due to the reassortant nature of rotavirus. The consensus sequences were aligned using the BioEdit program and then treated with the Fast PCR software to derive the primers. The derived primer sequences were submitted for a BLAST search to ensure alignment was exclusive to the desired target genes. The designed primers had specific bands and were efficient in detecting rotavirus in faecal samples than previously published primers. Thus, a successful surveillance of rotaviruses requires that primer pairs be updated regularly in order to detect the emergence of novel or “unusual types”, which have occurred by genetic drift causing nucleotide changes at the primer binding sites that result in typing failures. We recommend the use of the proposed primers in molecular surveillance studies for efficient detection of avian rotavirus.     

La Sota vaccination may not protect against virus shedding and the lesions of velogenic Newcastle disease in commercial turkeys

The aim of this project is to study the clinical signs and lesion of velogenic Newcastle disease (vND) in commercial turkeys, and also to find out if La Sota vaccination offered protection against these signs and lesions. The cockerels were included as positive controls. One hundred and twenty turkey poults and cockerels were divided into eight groups as follows: unvaccinated unchallenged turkeys (UUT), unvaccinated challenged turkeys (UCT), vaccinated unchallenged turkeys (VUT), vaccinated challenged turkeys (VCT), and along the same lines, the cockerel groups were UUC, UCC, VUC and vaccinated challenged cockerels (VCC). Vaccination was at 3 weeks of age while challenge was at 6 weeks of age. The unvaccinated turkeys and cockerels (UCT and UCC) showed different degrees of depression, diarrhoea and later paralysis at challenge. Total mortality was 100% in cockerels within 6 days, but 60% in turkeys. Similar but milder clinical signs were found in the VCC with a total mortality of 13.3%. The VCT showed mild drop in feed and water consumption, and no mortality. All the challenged groups had significant (p < 0.05) loss of weight when compared with their controls. Necropsy showed that while the UCC had severe proventricular haemorrhages, intestinal and caecal tonsil ulcers, the UCT had no digestive tract lesion. There was severe atrophy of the lymphoid organs in all the challenged groups. Histopathological sections of the bursa, spleen and thymus in all the challenged groups with special emphasis on the vaccinated and unvaccinated turkeys with mortalities of 0 and 60%, respectively, had very severe necrosis and depletion of the lymphoid tissue. Virus was isolated from the cloacal swabs. The haemagglutination inhibition antibodies were significantly higher (p < 0.05) in the challenged groups than the unchallenged. The above observations in the intestinal tracts of UCT are of diagnostic significance while the gross and microscopic lesions in the UCT and VCT show that La Sota vaccination may not protect turkeys against the destruction of the lymphoid organs by vND as earlier reported in chickens. This may lead to immunosuppression and production problems in areas where vND is enzootic.     

Virulence traits of avian pathogenic (APEC) and fecal (AFEC) <Emphasis Type="Italic">E. coli</Emphasis> isolated from broiler chickens in Algeria

Avian pathogenic E. coli (APEC) is the etiologic agent of avian colibacillosis, the most common disease responsible for chicken morbidity in the world. Although multiple virulence-associated factors were identified, their prevalence in Algeria is still poorly known. In the present research, 92 avian pathogenic E. coli (APEC) isolates were recovered from broilers with clinical signs and lesions of colibacillosis. In addition, 32 E. coli isolates collected from feces of healthy birds (AFEC) were included for comparison. All isolates were investigated by PCR for the presence of a total of 11 virulence-associated genes described for avian pathogenic (iroN, ompT, hlyF, iss, iutA, and fimC) and diarrheagenic E. coli (eae, stx, elt/est, ipaH, and aggR). The sensitivity of 39 APEC isolates to 16 antibiotics was also determined using antimicrobial pretreated microplates. Here, we report that 98% of the examined isolates host at least one of the tested virulence factors. The most prevalent genes in APEC were iutA (90.6%), ompT (86.9%), and iss (85.8%); whereas, iutA (78.1%), fimC (78.1%), and iroN (68.7%) were the highest prevalent genes in AFEC. Our data showed that none of the AFEC isolates harbor any of the tested diarrheagenic genes. Moreover, only elt/est (5.4%), stx (2.1%), and ipaH (2.1%) genes were carried by APEC isolates. We further established that ceftazodime, ceftiofur, mequindox, amoxicillin/clavulanic acid, and meropenem were the most efficient antibiotics against the analyzed APEC isolates. Overall, our findings provide more insights about APEC and AFEC virulence potential in Algeria which could participate in the fight against colibacillosis.     

Genomic characterization of the first oral avian papillomavirus in a colony of breeding canaries (<Emphasis Type="Italic">Serinus canaria</Emphasis>)

Papillomaviruses are non-enveloped, DNA viruses that infect skin and mucosa of a wide variety of vertebrates, causing neoplasias or simply persisting asymptomatically. Avian papillomaviruses, with six fully sequenced genomes, are the second most studied group after mammalian papillomaviruses. In this study, we describe the first oral avian papillomavirus, detected in the tongue of a dead Yorkshire canary (Serinus canaria) and in oral swabs of the same bird and other two live canaries from an aviary in Madrid, Spain. Its genome is 8,071 bp and presents the canonical papillomavirus architecture with six early (E6, E7, E1, E9, E2, E4) and two late open reading frames (L1 and L2) and a long control region between L1 and E6. This new avian papillomavirus L1 gene shares a 64% pairwise identity with FcPV1 L1, so it has been classified as a new species (ScPV1) within the Ethapapillomavirus genus. Although the canary died after showing breathing problems, there is no evidence that the papillomavirus caused those symptoms so it could be part of the oral microbiota of the birds. Hence, future investigations are needed to evaluate the clinical relevance of the virus.     

Early heat acclimation during incubation improves Japanese quail performance under summer conditions

Effects of exposing quail eggs to high temperature on the heat tolerance ability and productivity of birds were investigated. Four groups of 600 fertile eggs were randomly selected; the first group was incubated under 37.5 °C and the hatched chicks were reared under a gradual decrease in temperature from 35 to 24 °C (Control). The second group was exposed to 39.1 °C for 2 h/day during 4–14 days of embryogenesis and the hatched chicks were reared under a gradual decrease in temperature from 35 to 24 °C. The third group was incubated under 37.5 °C and the hatched chicks were exposed to 39?±?1 °C for 2 h/day during 4–14 days of age. The fourth group was exposed to 39?±?1 °C for 2 h during 4–14 days of embryogenesis and the hatched chicks were exposed to 39?±?1 °C for 2 h/day during 4–14 days of age. The temperature applied changed (P?<?0.01) embryo weight and incubation period. Birds exposed to high temperature during brooding had superior growth performance, dressed carcass, body temperature and health traits. Birds subjected to 39?±?1 °C during brooding exhibited decreased feed consumption and body weight gain. Finally, this work suggests that thermal acclimation during embryogenesis might offer a practical method for easing heat stress.

     

Separating component parts of soil respiration under <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> plantation in the Taihang Mountains,China

Partitioning the respiratory components of soil surface CO₂ efflux is important in understanding carbon turnover and in identifying the soil carbon sink/source function in response to land-use change. The sensitivities of soil respiration components on changing climate patterns are currently not fully understood. We used trench and isotopic methods to separate total soil respiration into autotrophic (R _A ) and heterotrophic components (R _H ). This study was undertaken on a Robinia pseudoacacia L. plantation in the southern Taihang Mountains, China. The fractionation of soil ¹³CO₂ was analyzed by comparing the δ¹³C of soil CO₂ extracted from buried steel tubes with results from Gas Vapor Probe Kits at a depth of 50 cm at the preliminary test (2.03‰). The results showed that the contribution of autotrophic respiration (fR _A ) increased with increasing soil depth. The contribution of heterotrophic respiration (fR _H ) declined with increasing soil depth. The contribution of autotrophic respiration was similar whether estimated by the trench method (fR _A , 23.50%) or by the isotopic method in which a difference in value of ¹³C between soil and plant prevailed in the natural state (RC, 21.03%). The experimental error produced by the trench method was insignificant as compared with that produced by the isotopic method, providing a technical basis for further investigations.     

Effect of FMD vaccination on semen quality parameters in Karan Fries and Murrah buffalo bulls

Effect of Foot and Mouth disease (FMD) vaccination was studied on semen quality parameters of 19 Karan Fries (KF) and eight Murrah (MU) breeding bulls during the period 2002 to 2004 at Artificial Breeding Complex, NDRI, Karnal. A total of non-vaccinated 155 KF and 72 MU bulls' ejaculates were taken as control, while 169 KF and 51 MU bulls' ejaculates, collected after vaccination, were used to study the effect of vaccination stress. The results showed that FMD vaccination had no significant (P > 0.05) effect on ejaculate volume and total volume per day of semen in both KF and MU bulls. Volume of semen increased slightly during post-vaccination period in both the breeds. After FMD vaccination, there was significant (P < 0.01) decrease in mass activity (2.27 ± 0.06 vs. 1.67 ± 0.07 and 2.49 ± 0.09. vs. 1.75 ± 0.10, for KF and MU, respectively), initial motility (56.89 ± 0.03% vs. 44.62 ± 0.02% and 62.26 ± 0.04% vs. 47.08 ± 0.05%, for KF and MU, respectively), sperm concentration (754.19 ± 23.96 vs. 554.14 ± 22.95 × 10⁶/ml and 848.61 ± 33.65 vs. 571.57 ± 39.99 × 10⁶/ml, for KF and MU, respectively), and total sperm output per ejaculate (3,685.94 ± 158.40 vs. 2,781.54 ± 151.70 × 10⁶ and 2,218.75 ± 133.14 vs. 1,582.84 ± 158.20 × 10⁶, for KF and MU, respectively). Application of FMD vaccine had significantly (P < 0.05) adverse effect on most of the seminal attributes during post-vaccination in KF and MU buffalo bulls. So, the spermiograms affected following vaccination suggest that in bovines, the semen collection and preservation should be suspended till normal fertility of sperm is restored to avoid the failure of conception from artificial insemination using such semen.     

Plasma pharmacokinetics and milk levels of ceftriaxone following single intravenous administration in healthy and endometritic cows

Pharmacokinetics and milk levels of ceftriaxone were studied in healthy and endometritic cows following single intravenous administration. The drug was detected up to 8 h of dosing in plasma of healthy and endometritic cows and the drug disposition followed three-compartment open model. The values of Vd_area, AUC, t_1/2β, Cl_B, MRT and P/C ratio were 0.50 ± 0.19 L.kg⁻¹, 62.2 ± 23.3 μg.ml⁻¹.h, 1.02 ± 0.07 h, 0.30 ± 0.09 L.kg⁻¹.h⁻¹, 1.55 ± 0.25 h and 0.52 ± 0.27, respectively, in healthy and 1.55 ± 0.52 L.kg⁻¹, 37.0 ± 17.1 μg.ml⁻¹.h, 1.56 ± 0.25 h, 0.56 ± 0.14 L.kg⁻¹.h⁻¹, 2.14 ± 0.34 h and 1.44 ± 0.60, respectively, in endometritic cows. The drug was detected in milk for 36 h after administration. For MIC₉₀ of 0.5 μg.ml⁻¹ the most appropriate dosage for ceftriaxone, would be 9.0 mg.kg⁻¹ repeated at 6 h intervals for the treatment of endometritis in cows.     

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.     

Study of male effect on feeding and estrus behavior of Afshari ewes

Tropical Animal Health and Production - This study was conducted to evaluate the male effect on the manifestation of estrus and feeding behavior of Afshari ewes during their breeding season. The...