Antibody detection-based differential ELISA for NDV-infected or vaccinated chickens versus NDV HN-subunit vaccinated chickens

With the advent of subunit vaccines for microbial diseases it is becoming increasingly important to be able to differentiate naturally infected animals from those vaccinated with the corresponding subunit vaccine. For avian viruses such as Newcastle disease virus (NDV), a whole virus-based ELISA cannot make such a differential diagnosis since in both cases the antisera would react with the whole virus. The nucleocapsid protein (NP) gene of the NDV Hitchner B1 strain was cloned, sequenced and expressed to develop a differential ELISA. The B1 NP had 95.7 and 96.1% amino acid identities with the NP of the d26 and Ulster 2C strains, respectively. The B1 NP expressed in a baculovirus expression vector (recNP) was the expected size and reacted with NDV-specific antibodies (Ab) in Western blots and by radioimmunoprecipitation. The ELISA using recNP-coated wells, tested on serum samples from flocks pretested with a commercial NDV kit gave results corresponding to those of the kit. Furthermore, use of both the renNP-based ELISA and a whole virus ELISA allowed the differentiation of birds vaccinated and a NDV haemagglutinin-neuraminidase (HN) expressing fowlpox virus from birds infected with NDV. This provides the basis for establishing an ELISA that discriminates between the antibody response to a recombinant fowlpox vaccine (expressing NDV HN protein) and that to live and inactivated NDV.     

Development of the simultaneous detection of Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus in potato tubers by a multiplex real-time PCR assay

Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum (Smith) Yabuuchi et al. race 3 are the causal agents of ring-rot and brown-rot of potato respectively. These diseases represent a serious threat to potato production in temperate climates. Both bacteria are listed as A2 pests in the EPPO region and as zero-tolerance quarantine organisms in the European Union. All the detection tests developed so far were only focused on the detection of a single pathogen while the absence of both bacteria has to be certified in the seed tubers. We have therefore developed a new multiplex real-time PCR assay to simultaneously detect both bacteria in a single assay. Additionally, the reliability of this molecular diagnostic test has been improved by the simultaneous amplification of an internal control, corresponding to a potato gene co-extracted from the sample. The polyvalence and the specificity of each set of bacterial primers and probes were evaluated on more than 90 bacterial strains. The limit of detection of this triplex real-time protocol was similar to those observed with other molecular protocols previously developed for the individual detection of one of these bacteria. A concordance of 100 % was obtained in a blind test mimicking the routine application of the technology. In conclusion, this new protocol represents a straightforward and convenient method potentially adapted to primary screening of potato tubers.     

Pharmacokinetics and bioavailability of doxycycline in fasted and nonfasted broiler chickens

The pharmacokinetics and the influence of food on the kinetic profile and bioavailability of doxycycline was studied after a single intravenous (i.v.) and oral dose of 10.0 mg/kg body weight in 7-week-old broiler chickens. Following i.v. administration the drug was rapidly distributed in the body with a distribution half-life of 0.21 +/- 0.01 h. The elimination half-life of 6.78 +/- 0.06 h was relatively long and resulted from both a low total body clearance of 0.139 +/- 0.007 L/h.kg and a large volume of distribution of 1.36 +/- 0.06 L/kg. After oral administration to fasted chickens, the absorption of doxycycline was quite fast and substantial as shown by the absorption half-life of 0.39 +/- 0.03 h, the maximal plasma concentration of 4.47 +/- 0.16 micrograms/mL and the time to reach the Cmax of 1.73 +/- 0.06 h. The distribution and the final elimination of the drug were slower than after i.v. administration. The absolute bioavailability was 73.4 +/- 2.5%. The presence of food in the intestinal tract reduced and extended the absorption (t1/2a = 1.23 +/- 0.21 h; Cmax = 3.07 +/- 0.23 micrograms/mL; tmax = 3.34 +/- 0.21 h). The absolute bioavailability was reduced to 61.1% +/- 4.4%.     

Protection of the nursing pig against experimentally induced enteric colibacillosis by vaccination of dam with fimbrial antigens of E coli (K88, K99 and 987P)

Pregnant gilts were vaccinated with two doses of alhydrogel adsorbed fimbrial antigens of Escherichia coli (K88ab, K88ac, K99 and 987P) supplemented with beta toxoid of Clostridium perfringens type C. Their piglets, and piglets of nonvaccinated gilts, were subsequently orogastrically challenged with one or other of the four fimbrial types of enteropathogenic E coli. Some of the vaccinated animals were reinjected with a single dose of the vaccine during second gestation and their piglets, and piglets of non-vaccinated sows, were challenged the same way as were litters of gilts. Blood serum and colostra were examined for antibodies to the four fimbrial antigens of E coli and for antitoxin to beta toxin of C perfringens type C. It was found that: (1) a highly significant reduction in mortality and morbidity was achieved in vaccinated litters against all four challenge strains of E coli; (2) excretion of K88ab and K88ac but not of K99 and 987P challenge strains was significantly reduced; (3) revaccination of sows by a single dose of the vaccine during second gestation conferred complete protection against mortality and highly significant protection against morbidity; (4) no correlation was noted between colostral or seroagglutinins to fimbrial antigens of E coli and mortality rates in litters challenged with homologous fimbrial types of E coli, but good correlation was found between colostral precipitins to K88 antigens and mortality rates in litters; (5) antitoxin value in 97 per cent of colostrum of vaccinated sows was 10 iu equivalent of C perfringens type C toxin or more per ml of colostrum.     

Diffusion of radiodense contrast medium after perineural injection of the palmar digital nerves

Limited information exists on distribution of local anaesthetic solution following palmar digital nerve blocks. The aim of this study was to demonstrate potential distribution of local anaesthetic solution following perineural injection of the palmar digital nerves using 2 different volumes of contrast medium and 2 different injections sites. Twelve mature horses were used. Perineural injection of the palmar digital nerves were performed at the level of or 2 cm proximal to the proximal aspect of the ipsilateral ungular cartilage, using 1.5 or 2.5 ml radiopaque contrast medium. In total, 96 injections were performed. Four standard radiographic views of the pastern were obtained immediately after injections and 10 and 20 min later. Images were analysed subjectively and objectively. After distal injections, the contrast medium was more localised around the injection site; after proximal injections the contrast patch had greater proximal–distal length. The greatest proximal diffusion was to 31.7% of the length of the proximal phalanx (from the level of the proximal interphalangeal joint) after distal injections and to 70% after proximal injections. The larger volume resulted in significantly greater proximal diffusion than the smaller volume at the distal, but not at the proximal injection site (P<0.01). There was significant proximal diffusion with time after proximal and distal injections (P<0.01). In most limbs, numerous radiopaque lines of various thickness extended proximally from the contrast patches; subjectively, their number and thickness were greater at the distal injection site. In conclusion, palmar digital nerve blocks at the level of the ungular cartilage using ≤2.5 ml local anaesthetic solution may improve proximal interphalangeal joint and pastern region pain. If using a more proximal site, distal fetlock region pain may be improved. Due to diffusion into lymphatic vessels, too small a volume at the distal injection site may not provide sufficient analgesia.     

Tree-Based Methods as an Alternative to Logistic Regression in Revealing Risk Factors of Crib-Biting in Horses

Determining the risk factors might help in designing prevention of crib-biting. Logistic regression is a commonly used statistical method for finding risk factors, but tree-based methods are also getting more popular. An important difference between these two statistical approaches is that logistic regression makes a number of assumptions about the underlying data, whereas tree-based methods do not. Another difference is that logistic regression can be used to derive odds ratios for the significant risk factors, whereas tree-based methods create a tree where the ramifications represent the risk factors. The probability of occurrence is assigned to each end of branch in the tree. Data of horses used for noncompetition purposes were analyzed with three statistical approaches: logistic regression, classification tree, and conditional inference tree methods. By this, we compared the advantages and disadvantages of these statistical methods. No difference was found between the two tree-based methods regarding the structure and prediction accuracy of the trees. Compared to them, logistic regression revealed fewer risk factors, and also the number of the stereotypic horses classified correctly by the model was less. The representation of the tree-based methods is closer to medical reasoning and also high-order interaction of the risk-factors can easily be visualized. Our results suggest that tree-based methods can be a new alternative in revealing risk factors, even if used alone or together with logistic regression.     

Study of vertical transmission of fowl adenoviruses

The vertical transmission of fowl adenoviruses (FAdVs) was studied by polymerase chain reaction (PCR) and virus isolation. Liver, spleen, kidney, and bursa of Fabricius were collected from 60 chicks 1 d old representing progenies hatched to 6 broiler breeder flocks in 6 geographically different premises in Ontario, Canada. The presence of FAdV DNA sequences was detected by PCR with the use of primers specific for the conserved pVI gene of FAdV-9 in 58 (24%) of the 240 samples tested. All samples from 1 flock were negative for FAdV sequences, and only a few samples were positive in 3 flocks, whereas 32% and 72% of the samples from the other 2 flocks were positive. Testing of 1 sample with primers designed to amplify the L1 region of the hexon protein gene and amino acid sequence analysis of the PCR product indicated that the sequences were similar to serotype-8a FAdV sequences. No fowl adenoviruses were isolated in chicken hepatoma cells from any of the 30 samples inoculated. These findings imply that vertical transmission and establishment of latent infection with FAdVs can occur in chickens.     

Effects of Foliar Biofertilizer Sprays on Nutrient Uptake,Yield, and Quality Parameters of Blaufrankish (Vitis vinifera L.) Grapes

Nowadays the main task of scientists is to find natural ways of improving plant productivity that lead to environmentally friendly agriculture. Biofertilizers have a great potential to achieve this aim but unfortunately there is little information about their application in grape growing in Hungary. For this reason, a foliar nutrition experiment was conducted to investigate the effect of two biofertilizers (an algae product and a biostimulator, containing amino acids) on yield, leaf nutrient concentration, and quality parameters of Blaufrankish grape variety. The study was conducted in 2012 at Noszvaj in northeastern Hungary in a 24-year-old grapevine plantation on cv. Blaufrankish. Treatments (application time and doses) were adjusted to the phenological phases of grapevine. Effect of treatments was monitored by soil and leaf analysis, grape quality measurements, and field observations. Treatments increased the yield but did not affect the fruit quality compared to the control. The applied products significantly increased the bunch weight and the size of berries. Applied biofertilizers had no effect on leaf nutrient concentration. According to our field observations, it seemed that treated vines had larger and greener leaves.     

Pathogenicity of infectious bronchitis virus isolates from Ontario chickens

Infectious bronchitis (IB) is one of the important viral diseases of chickens, and in spite of regular vaccination, IB is a continuous problem in Canadian poultry operations. In an earlier study using sentinel chickens we determined the incidence of infectious bronchitis virus (IBV) in Ontario commercial layer flocks. The objective of this study was to determine the pathogenicity of 5 nonvaccine-related IBV isolates recovered from the sentinel birds. The clinical signs, gross, and histological lesions in specific pathogen-free chickens indicated that all 5 isolates caused mild lesions in the respiratory tract. An important finding of this study was the significantly lower average daily weight gain among virus-inoculated groups of chickens during the acute phase of infection. Based on sequences of part of the S1 gene IBV-ON2, IBV-ON3, and IBV-ON5 formed a cluster and they were closely related to strain CU-82792. IBV-ON4 had 98.7% identity with the strain PA/1220/9, a nephropathogenic variant.