In vitro effects of individual fatty acids on protozoal numbers and on fermentation products in ruminal fluid from cattle fed a high-concentrate, barley-based diet

The objective of this study was to investigate the effects of sodium salts of individual fatty acids on protozoal numbers and ruminal fermentation variables in vitro. Ruminal inoculum was obtained from two heifers fed a finishing diet consisting of (DM basis) 90% rolled barley grain, 4% barley silage, 5% soybean meal, and 1% mineralized salt. Fatty acids (FA) were included individually in the inoculum as follows: C6:0, C8:0, and C10:0 at concentrations (wt/vol) of 0.0625, 0.125, and 0.25%; C14:0 and C18:0 at concentrations of 0.125, 0.25, and 0.5%; and C12:0, C16:0, C18:1, C18:2, and C18:3 at concentrations of 0.25, 0.5, and 1.0%. 15N-Labeled casein was included as a N tracer. In the presence of medium-chain saturated FA (particularly C10:0 and C12:0), no ciliate protozoa (99.8%Entodinium spp.) were recovered from the incubation medium. Long-chain unsaturated FA (C18:3, C18:2, C18:1) also decreased (P < 0.05) protozoal numbers. At all concentrations tested, C10:0 and C12:0 decreased (P < 0.05) ammonia and total VFA concentrations (by 29 and 22%, respectively) and increased (P < 0.05) concentrations of total free amino acids, reducing sugars, and soluble protein. At the greatest concentrations of these FA, xylanase and amylase activities of the incubation media were decreased (P < 0.05). The C18 unsaturated FA increased (P < 0.05) the polysaccharide-degrading activities of the media. These in vitro results suggest that long-chain unsaturated FA in combination with medium-chain saturated acids have the potential to decrease protozoal numbers and ruminal ammonia utilization in cattle fed high-grain diets.     

Biological and molecular characterization of chicken anemia virus isolates from Slovenia

The presence of chicken anemia virus (CAV) in Slovenia was confirmed by inoculation of 1-day-old chickens without antibodies against CAV and isolation of the virus on the Marek's disease chicken cell-MSB1 line and by polymerase chain reaction (PCR). Experimental inoculation of 1-day-old chickens resulted in lower hematocrit values, atrophy of the thymus, and atrophy of bone marrow. CAV was confirmed by PCR in the thymus, bone marrow, bursa of Fabricius, liver, spleen, ileocecal tonsils, duodenum, and proventriculus. The nucleotide sequence of the whole viral protein (VP)1 gene was determined by direct sequencing. Alignment of VP1 nucleotide sequences of Slovenian CAV isolates (CAV-69/00, CAV-469/01, and CAV-130/03) showed 99.4% to 99.9% homology. The VP1 nucleotide sequence alignment of Slovenian isolates with 19 other CAV strains demonstrated 94.4% to 99.4% homology. Slovenian isolates shared highest homology with the BD-3 isolate from Bangladesh. Alignment of the deduced VP1 amino acids showed that the Slovenian isolates shared 100% homology and had an amino acid sequence most similar to the BD-3 strain from Bangladesh (99.6%) and were 99.1% similar to the G6 strain from Japan and the L-028 strain from the United States. The Slovenian isolates were least similar (96.6%) to the 82-2 strain from Japan. A phylogeneric analysis on the basis of the alignment of the VP1 amino acids showed that CAV isolates used in the study formed three groups that indicated the possible existence of genetic groups among CAV strains. The CAV isolates were grouped together independent of their geographic origin and pathogenicity.     

IS900-PCR-based detection and characterization of Mycobacterium avium subsp. paratuberculosis from buffy coat of cattle and sheep

Johne's disease is one of the main causes of economic losses in ruminants and a major health hazard in the developing and developed world. Up till now, many microbiological, serological and molecular methods have been tried for the detection of Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we attempt a PCR-based detection of IS900, distinct insertion sequences of MAP from the buffy coat of cattle (n=262) and sheep (n=78), and direct genotyping by single strand conformational polymorphism (SSCP). A total of 30 (11.45%) cattle and one sheep (1.28%) were positive for MAP-IS900. This IS900-based PCR detection proved highly specific, particularly when tested on other non-MAP strains. SSCP analysis grouped the MAP-IS900 into four distinct clusters based on different band patterns. Nucleotide sequence variability between MAPs detected from sheep (GenBank accession ) and cattle (GenBank accession -) was noticed in the study. Although, in recent years IS900-PCR-based detection of MAP from WBCs is being used in human, its use in animals is still limited. Our work not only supports its use in animals but also suggests further IS900-SSCP-based MAP-genotyping, coupled with DNA sequencing, as a promising tool for rapid and effective Johne's disease surveillance.     

Molecular detection of BHV-1 in artificially inoculated semen and in the semen of a latently infected bull treated with dexamethasone

Two polymerase chain reaction (PCR) assays specific for glycoprotein B (gB) and glycoprotein E (gE) gene detection, respectively, were adopted for the detection of bovine herpesvirus-1 (BHV-1) in naturally infected bulls. The methods were tested on bovine semen artificially inoculated with BHV-1 and were compared with an optimised virus isolation method. Raw and extended semen samples were diluted in minimal essential medium (MEM) and spiked with equal dose of BHV-1. The extended semen was found to be more toxic for the cells than the raw semen, while the viral DNA could be detected by the PCR method in all tested dilutions of raw and extended semen samples. The sensitivity of both methods was compared also for BHV-1 detection in semen, nasal swabs and leucocytes of a seropositive bull in a different time period after virus reactivation with dexamethasone treatment. The sensitivity of virus detection by the PCR method was equivalent to that of virus isolation in cell culture. However, PCR was shown to be faster and easier to perform and may be a good alternative to virus isolation especially when bovine semen has to be screened for BHV-1 prior to artificial insemination.     

The area under the disease progress stairs: calculation, advantage, and application

The area under the disease progress curve (AUDPC) is frequently used to combine multiple observations of disease progress into a single value. However, our analysis shows that this approach severely underestimates the effect of the first and last observation. To get a better estimate of disease progress, we have developed a new formula termed the area under the disease progress stairs (AUDPS). The AUDPS approach improves the estimation of disease progress by giving a weight closer to optimal to the first and last observations. Analysis of real data indicates that AUDPS outperforms AUDPC in most of the tested trials and may be less precise than AUDPC only when assessments in the first or last observations have a comparatively large variance. We propose using AUDPS and its standardized (sAUDPS) and relative (rAUDPS) forms when combining multiple observations from disease progress experiments into a single value.     

Molecular characterization of Pseudomonas syringae isolates from fruit trees and raspberry in Serbia

Infection of fruit trees by Pseudomonas syringae is a potentially serious problem that may limit the establishment and sustained productivity of pome and stone fruit orchards in Serbia. To estimate possible diversity of Pseudomonas syringae fruit trees strains, we collected a set of strains in several areas of Serbia. The samples were taken from infected orchards with raspberry, plum, cherry, sour cherry, peach, pear and apple trees. Genetic diversity of P. syringae strains isolated from fruit trees was determined by using SpeI macrorestriction analysis of genomic DNAs by pulsed-field gel electrophoresis (PFGE) and REP-PCR. Molecular analysis showed that most of isolates had unique profiles, with the exception of isolates from plum and cherry that displayed profiles identical to each other and similar to P. syringae pv. morsprunorum. The study presented here clearly demonstrates the discriminative power of molecular techniques in enabling a detailed analysis of the genetic variations between strains of P. syringae from different pome and stone fruit hosts in Serbia.     

Leukocytic responses and intestinal mucin dynamics of broilers protected with Enterococcus faecium EF55 and challenged with Salmonella Enteritidis

The protective effect of Enterococcus faecium EF55 in chickens challenged with Salmonella enterica serovar Enteritidis phage type 4 (SE PT4) was assessed. The antibacterial effect on the bacterial microflora in the small intestine in relation to white blood cell count, phenotyping of peripheral blood and intestinal lymphocytes, functional activity of lymphocytes and phagocytes and mucin quantitation were investigated. Day-old chicks (85) were randomly divided into four groups. The probiotic group (EF) and Salmonella + probiotic group (EFSE) received E. faecium EF55 (10⁹ CFU – 3 g/group/day) for 21 days. The Salmonella group (SE) and EFSE group were infected with Salmonella Enteritidis (10⁸ CFU in 0.2 ml PBS) in a single dose per os on day four of the experiment. The control group chicks (C) were fed a commercial diet without added bacteria. Supplementation of EF55 in the diet of the chickens in the EFSE group, challenged with S. Enteritidis, caused the density of the intestinal mucin layer to increase significantly in non-specific regions (duodenum and jejunum), but decrease significantly in target regions (caeca) for S. Enteritidis. Probiotic treatment also appeared to result in a significantly higher number of lymphocytes in peripheral blood and a tendency to increase CD3, CD4, CD8, and IgM positive cells 3 days post-infection with S. Enteritidis. The results demonstrated an antibacterial effect and suggested that EF55 had a moderating effect on intestinal mucin production and leukocytic response in the early phase of S. Enteritidis infection.     

Optimizing early detection of avian influenza H5N1 in backyard and free-range poultry production systems in Thailand

For infectious diseases such as highly pathogenic avian influenza caused by the H5N1 virus (A/H5N1 HP), early warning system is essential. Evaluating the sensitivity of surveillance is a necessary step in ensuring an efficient and sustainable system. Stochastic scenario tree modeling was used here to assess the sensitivity of the A/H5N1 HP surveillance system in backyard and free-grazing duck farms in Thailand. The whole surveillance system for disease detection was modeled with all components and the sensitivity of each component and of the overall system was estimated. Scenarios were tested according to selection of high-risk areas, inclusion of components and sampling procedure, were tested. Nationwide passive surveillance (SSC1) and risk-based clinical X-ray (SSC2) showed a similar sensitivity level, with a median sensitivity ratio of 0.96 (95% CI 0.40-15.00). They both provide higher sensitivity than the X-ray laboratory component (SSC3). With the current surveillance design, the sensitivity of detection of the overall surveillance system when the three components are implemented, was equal to 100% for a farm level prevalence of 0.05% and 82% (95% CI 71-89%) for a level of infection of 3 farms. Findings from this study illustrate the usefulness of scenario-tree modeling to document freedom from diseases in developing countries.