Use of serology and real time PCR to control an outbreak of bovine brucellosis at a dairy cattle farm in the Nile Delta region,Egypt

Background

Bovine brucellosis remains one of the most prevalent zoonotic infections affecting dairy cattle in developing countries where the applied control programs often fail. We analyzed the epidemiologic pattern of bovine brucellosis in a dairy cattle herd that showed several cases of abortions after regular vaccination with RB51 (B. abortus vaccine). In 2013 thirty dairy cows, from a Holstein-Friesian dairy herd with a population of 600 cattle, aborted five months post vaccination by a regular RB51 vaccine. Blood samples were drawn from milking cows and growing heifers, as well as heifers and cows pregnant up to 6 months. These samples were collected in June 2013 (n?=?257) and May 2014 (n?=?263) and were tested by real time (rt)-PCR as well as serological tests, in particular Rose Bengal Test (RBT), Enzyme-Linked Immunosorbent Assays (ELISA) and Fluorescence Polarization Assay. Tissue specimens were also collected from an aborted fetus and cultured. Isolates were subjected to bacteriological typing tests at the genus and species levels.

Results

Five months post vaccination with RB51 vaccine, Brucella (B.) DNA was detected in blood samples of cows by rt-PCR. The serological tests also revealed the spread of Brucella field strains within the herd in 2013. Four Brucella isolates were recovered from specimens collected from the aborted fetus. These isolates were typed as follows: one B. abortus RB51 vaccine strain and three isolates of B. abortus field strain. The seropositive cows with positive rt-PCR might indicate an infection by the Brucella field strain; while the positive rt-PCR results from seronegative animals may either be due to circulating RB51 vaccine DNA in vaccinated animals or to circulating field strain in infected animals before seroconversion.

Conclusion

The results herein suggest that PCR can be a good supplementary tool in an outbreak situation, if an assay is available that can differentiate vaccine and field strains with a high analytical sensitivity. We recommend using RBT and ELISA in parallel in outbreak situations, to identify as many infected animals as possible during the initial screenings. This test procedure should be repeated for at least three successive negative tests, with one month interval.

     

Methane emissions and δ13C composition from beef steers consuming increasing proportions of sericea lespedeza hay on bermudagrass hay diets

An experiment was conducted to evaluate the effects of different proportions of ‘Au Grazer’ sericea lespedeza [SL, Lespedeza cuneata (Dum. Cours.) G. Don], a legume rich in condensed tannins (CT), on nutrient intake and digestibility, and to estimate methane (CH₄) emissions and ¹³C isotopic composition (δ¹³C_CH4) from beef steers consuming a forage-based diet. Twenty-five Angus-crossbred steers were distributed in a randomized complete block design (344 ± 48 kg initial BW), and randomly assigned to one of five treatments: 0SL, 25SL, 50SL, 75SL, and 100SL, diets containing 0%, 25%, 50%, 75%, and 100% of SL hay, respectively, mixed with ‘Tifton-85’ bermudagrass hay (Cynodon spp.). The study was carried out for two experimental periods of 21-d each. The statistical model included the fixed effect of treatment and random effects of block, experimental period, and their interaction. Apparent total tract digestibility of crude protein, neutral detergent fiber, and acid detergent fiber was linearly decreased (P < 0.001) by the inclusion of SL. No effects were observed for total CH₄ emissions per day, nor for CH₄ relative to organic matter intake or digestible organic matter with the inclusion of SL. However, emission of CH₄ in relation to intake of CT was affected by treatment (P < 0.001). A linear (P < 0.001) decrease and a quadratic effect (P < 0.001) were observed for δ¹³C of diets and gas, respectively, in which diets and enteric CH₄ with greater inclusion of SL were more depleted in ¹³C. Moreover, the difference in δ¹³C between diets and gas (Δδ¹³C) had a linear decrease (P = 0.001) with the inclusion of SL. The model developed to predict the C₃ proportions in the enteric CH₄ fitted to predicted values (P < 0.0001). Therefore, greater proportions of SL resulted in lesser CH₄ emission when CT intake was considered and the isotopic composition from enteric CH₄ was able to predict the contribution of SL in the emissions.     

Changes in free amino acids and sugars in potatoes due to sulfate fertilization and the effect on acrylamide formation

To examine how sulfur deprivation may affect acrylamide formation in cooked potatoes, three varieties of potato were grown under conditions of either severe sulfur deprivation or an adequate supply of sulfur. In all three varieties sulfur deprivation led to a decrease in acrylamide formation, even though the levels of sugars, which are acrylamide precursors, were higher in tubers of the sulfur-deprived plants. In one variety the concentration of free asparagine, the other precursor for acrylamide, was also higher. There was a very close correlation between the concentration of asparagine in the tubers expressed as a proportion of the total free amino acid pool and the formation of acrylamide upon cooking, whereas sugars were poorly correlated with acrylamide. In potatoes, where concentrations of sugars are usually limiting, competition between asparagine and other amino acids participating in the Maillard reaction may be a key determinant of the amount of acrylamide that is formed during processing.     

Determination of the crop coefficient for grafted ‘Tahiti’ lime trees and soil evaporation coefficient of Rhodic Kandiudalf clay soil in Sao Paulo,Brazil

The expansion of permanent trickle irrigation systems in Sao Paulo (Brazil) citrus has changed the focus of irrigation scheduling from determining irrigation timing to quantifying irrigation amounts. The water requirements of citrus orchards are difficult to estimate, since they are influenced by heterogeneous factors such as age, planting density and irrigation system. In this study, we estimated the water requirements of young ‘Tahiti’ lime orchards, considering the independent contributions from soil evaporation and crop transpiration by splitting the crop coefficient (Kc = ETc/ETo) into two separate coefficients; Ke, a soil evaporation coefficient and Kcb, a crop transpiration coefficient. Hence, the water requirement in young ‘Tahiti’ lime (ET_y) is ET_y = (Ke + Kcb) · ETo, where ETo is the reference crop evapotranspiration. Mature tree water requirement (ET_m) is ET_m = Kcb · ETo, assuming no soil water evaporation. Two lysimeters were used; one was 1.6 m in diameter and 0.7 m deep, and the other was 2.7 m in diameter and 0.8-m deep. The first one was used to calculate evaporation and the second one was used for transpiration. ETo was estimated by the Penman–Monteith method (FAO-56). The measurements were conducted during a period between August 2002 and April 2005 in Piracicaba, Sao Paulo state, Brazil. The lysimeters were installed at the center of a 1.0-ha plot planted with ‘Tahiti’ lime trees grafted on ‘Swingle’ citrumelo rootstock. The trees were 1-year old at planting, spaced 7 × 4 m, and were irrigated by a drip irrigation system. During the study period, Kc varied between 0.6 and 1.22, and Kcb varied between 0.4 and 1.0. The results suggested that for young lime trees, the volume of water per tree calculated by Ke + Kcb is about 80% higher than the volume calculated using Kc. For mature trees, the volume of water per tree calculated using just Kcb can be 10% less than using Kc. The independent influence of soil evaporation and transpiration is important to better understand the water consumption of young lime trees during growth compared to mature lime trees.     

An enrichment microsphere immunoassay for the detection of <Emphasis Type="Italic">Pectobacterium atrosepticum</Emphasis> and <Emphasis Type="Italic">Dickeya dianthicola</Emphasis> in potato tuber extracts

An enrichment microsphere immunoassay (MIA) was developed, based on the Luminex xMAP® technology, for the simultaneous (duplex) detection of Pectobacterium atrosepticum (former name Erwinia carotovora subsp. atroseptica) (Pca) and Dickeya dianthicola (former name Erwinia chrysanthemi) (Dcd) in potato plant extracts. Target bacteria in the extracts were enriched for 48 h in a semi-selective broth containing polypectate under low oxygen conditions. Samples were subsequently incubated with antibody-coated colour-coded microspheres (beads) and with secondary antibodies conjugated with Alexa Fluor® 532, a reporter dye. Samples were analyzed with the Luminex analyzer, in which one laser identified each microsphere and another laser the reporter dye conjugated to the secondary antibodies. The assay required minimal sample preparation, could be completed in 1 h, was performed in 96 wells microtitreplates and required no wash steps. The limit of detection for the duplex enrichment MIA was 100–1000 cfu ml^?1, which was a hundred times lower than of an enrichment-ELISA. Without enrichment, the sensitivity of MIA and ELISA was largely similar and ranged between 10⁶ and 10⁷ cells ml^?1. No difference in sensitivity was found between a MIA in a single or duplex format. In a comparative test with non-infected potato plant extracts and extracts from plants infected with Pca or Dcd, results of the enrichment MIA correlated well with those of the enrichment ELISA and enrichment PCR. These results indicate that MIA can be reliably used for multiplex detection of soft rot Enterbacteriaceae in crude potato plant extracts. The technology is an attractive and cost-effective alternative to other detection methods, including ELISA.     

Gross nitrogen mineralisation and fungi-to-bacteria ratios are negatively correlated in boreal forests

In terrestrial ecosystems, gross nitrogen mineralisation is positively correlated to microbial biomass but negatively to soil organic matter C-to-N ratios; the influence of the microbial community structure is less well known. Here, we relate rates of gross N mineralisation to fungi-to-bacteria ratios in three natural forest types of contrasting N availability and in a long-term N-loading experiment in a boreal forest. We report, for the first time, a strong negative correlation between gross N mineralisation and the fungi-to-bacteria ratio ( = 0.91, P = 0.0005, N = 7). There was also a negative correlation between gross N mineralisation and the C-to-N ratio ( = 0.89, P = 0.001, N = 7), but a weaker positive correlation between gross N mineralisation and soil pH ( = 0.64, P = 0.019, N = 7). Our analysis suggests that soil fungi-to-bacteria and C-to-N ratios are interrelated and that they exert strong influences on soil N cycling in boreal forests.     

Autoimmune hepatitis-specific antibodies against soluble liver antigen and liver cytosol type 1 in patients with chronic viral hepatitis

Background

Non-organ specific autoantibodies are highly prevalent in patients with chronic hepatitis C (HCV). Among them, anti-liver kidney microsomal type 1 (LKM1) antibody – the serological marker of type 2 autoimmune hepatitis (AIH-2)- is detected in up to 11% of the HCV-infected subjects. On the other hand, anti-liver cytosol type 1 antibodies (anti-LC1) – either in association with anti-LKM1, or in isolation- and anti-soluble liver antigen antibodies (anti-SLA) have been considered as useful and specific diagnostic markers for AIH. However, their specificity for AIH has been questioned by some recent studies, which have shown the detection of anti-LC1 and anti-SLA by immunoprecipitation assays in HCV patients irrespective of their anti-LKM1 status. The aim of the present study was to test the anti-LC1 and anti-SLA presence by specific enzyme linked immunosorbent assays (ELISAs), in a large group of Greek HCV-infected patients with or without anti-LKM1 reactivity as firstly, immunoprecipitation assays are limited to few specialized laboratories worldwide and cannot be used routinely and secondly, to assess whether application of such tests has any relevance in the context of patients with viral hepatitis since antibody detection based on such ELISAs has not been described in detail in large groups of HCV patients.

Methods

One hundred and thirty eight consecutive HCV patients (120 anti-LKM1 negative and 18 anti-LKM1 positive) were investigated for the presence of anti-LC1 and anti-SLA by commercial ELISAs. A similar number (120) of chronic hepatitis B virus (HBV) infected patients seronegative for anti-LKM1 was also tested as pathological controls.

Results

Six out of 18 (33%) anti-LKM^pos/HCV^pos patients tested positive for anti-LC1 compared to 1/120 (0.83%) anti-LKM^neg/HCV^pos patients and 0/120 (0%) of the anti-LKM1^neg/HBV^pos patients (p < 0.001 for both comparisons). Anti-SLA antibodies were not present in any of the HCV (with or without anti-LKM1) or HBV-infected patients.

Conclusion

We showed that anti-LC1 and anti-SLA autoantibodies are not detected by conventional assays in a large group of anti-LKM1 negative patients with chronic hepatitis B and C infections. Based on these results we cannot find any justification for the application of anti-LC1 and anti-SLA tests in the routine laboratory testing of viral hepatitis-related autoantibody serology with the only potential exception being the anti-LC1 screening in anti-LKM1^pos/HCV^pos patients.

     

Effect of fibers on the flow property of turbulent fiber suspensions in a contraction

In the flow of turbulent fiber suspensions flowing through a contraction with rectangular cross-section, the Reynolds averaged Navier-Stokes equation with the term of additional stress resulting from fibers was solved with the Reynolds stress model to get distributions of the mean velocity, mean pressure, turbulent kinetic energy and turbulent dissipation. It is found that the mean velocities at exit are small around the center and large near the wall for higher concentration. Fibers reduce turbulent intensity and turbulent dissipation at central line, but enhance them over the cross section at exit. Fibers have no effect of restraint on the turbulence in the contraction flow. The additional stress resulting from fibers plays a role in the increase of drag.     

Chemical Composition of Green Tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis>) Infusions Commercialized in Portugal

To evaluate the potential benefits and risks associated with tea consumption it is important to identify the constituents of this beverage. Levels of some minerals, caffeine and catechins in green tea samples commercialized in Portugal were evaluated. Potassium is the metal present in larger amount (92–151 mg/l). The content of sodium, calcium, fluoride, aluminium, manganese and iron were 35–69, 1.9–3.5, 0.80–2.0, 1.0–2.2, 0.52–1.9, 0.020–0.128 mg/l, respectively. Chromium and selenium were not detected. The resulting data showed considerable variability in catechins content. The levels of epigallocatechin gallate (EGCG) ranged from 117 to 442 mg/l, epicatechin 3-gallate (EGC) from 203 to 471 mg/l, epigallocatechin (ECG) from 16.9 to 150 mg/l, epicatechin (EC) from 25 to 81 mg/l and catechin (C) from 9.03 to 115 mg/l. Caffeine contents in the green tea infusions studied were between 141–338 mg/l. Green tea infusions provide significant amounts of catechins and could be an important source of some minerals.