Tick-borne fever caused by Anaplasma phagocytophilum in Germany: first laboratory confirmed case in a dairy cattle herd

Four cows from North-West Germany have been diagnosed with tick-borne fever (TBF) based on the demonstration of morulae in neutrophilic granulocytes in their blood smears, positive signals in real-time PCR specific for Anaplasma phagocytophilum using DNA extracted from their buffy coats, and demonstration of specific antibodies in their sera using a commercially available immunofluorescence assay. Clinical findings included high fever, decreased milk production, lower limb edema with stiff walking, eye and nasal discharge, and depression. These signs developed about a week after the animals had been brought to the pasture for the first time in their life. All cows recovered after 5-15 days, although DNA of A.phagocytophilum could be detected by real-time PCR up to 6 weeks after onset of the disease. Considering the known prevalences of A.phagocytophilum in ticks in Germany and its detection in dogs and horses, we think that underdiagnosing of TBE in cattle is highly likely. Therefore TBF should be taken into account as differential diagnosis in case of high fever and/or a sudden decrease in milk production in pastured animals.     

A Universal `One-tube' RT-PCR Protocol for Amplifying Isolates of Bovine Viral Diarrhoea Virus

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E ^RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10⁶ infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.     

A single receptor binds both insulin-like growth factor II and mannose-6-phosphate

Amino acid sequences deduced from rat complementary DNA clones encoding the insulin-like growth factor II (IGF-II) receptor closely resemble those of the bovine cation-independent mannose-6-phosphate receptor (Man-6-P receptorCI), suggesting they are identical structures. It is also shown that IGF-II receptors are adsorbed by immobilized pentamannosyl-6-phosphate and are specifically eluted with Man-6-P. Furthermore, Man-6-P specifically increases by about two times the apparent affinity of the purified rat placental receptor for 125I-labeled IGF-II. These results indicate that the type II IGF receptor contains cooperative, high-affinity binding sites for both IGF-II and Man-6-P-containing proteins.     

Association and General Notes

In a field disease outbreak, 60 female goats died over a 2–3 week period. Necropsies of seven of these does revealed that six had an acute exudative necrotising broncho-pneumonia, and moderate to high numbers of Pasteurella haemolytica were isolated from their lungs. Caprine herpesvirus, identified as Bovid herpesvirus type 6, was isolated from the lungs of two of these does, including one with pneumonia, and from nasal swabs of in-contact goats. Clinical disease was only observed in does, and deaths began 3 weeks after the introduction of a mob of goat hoggets from another farm.     

Virus Detection in Questing Ticks is not a Sensitive Indicator for Risk Assessment of Tick‐Borne Encephalitis in Humans

Tick‐borne encephalitis virus (TBEV) is the most important tick‐transmitted arbovirus causing human disease in Europe, but information on its endemic occurrence varies between countries because of differences in surveillance systems. Objective data are necessary to ascertain the disease risk for vaccination recommendations and other public health interventions. In two independent, separately planned projects, we used real‐time RT‐PCR to detect TBE virus in questing ticks. In Poland, 32 sampling sites were selected in 10 administrative districts located in regions where sporadic TBE cases were reported. In Germany, 18 sampling sites were selected in two districts located in a region with high TBE incidence. Altogether, >16 000 ticks were tested by real‐time RT‐PCR, with no sample testing positive for TBEV. A systematic search for published studies on TBEV prevalence in ticks in Poland and Germany also suggested that testing large numbers of collected ticks could not consistently assure virus detection in known endemic foci. Although assignment of results to administrative regions is essential for TBE risk mapping, this was possible in only 10 (investigating 22 417 ticks) of 15 published studies (>50 000 ticks) identified. We conclude that the collection and screening of ticks by real‐time RT‐PCR cannot be recommended for assessment of human TBE risk. Alternative methods of environmental TBEV monitoring should be considered, such as serological monitoring of rodents or other wildlife.     

Apparent digestibility coefficients of feed ingredients and their predictability in compound diets for gilthead seabream, Sparus aurata L.

Apparent digestibility of crude protein, amino acids, lipid, carbohydrate and energy was measured for a range of feed ingredients fed to gilthead seabream, Sparus aurata L. — fish meal, poultry meal, meat meal, blood meal, squid meal, extracted soyabean and wheat flour. Chromic oxide was used as a non-absorbed reference substance and faeces were collected by stripping. Diets compounded from mixtures of these ingredients were then used to examine the possibility of predicting the digestibility of formulated diets.
Apparent digestibility of crude protein ranged from 79% to 90%, lipids from 83% to 95% and energy from 72% to 88% in the different ingredients. Apparent digestibility of carbohydrates was lower and ranged from 49% to 77%. Apparent digestibility of amino acids was higher than that of crude protein and differences were found among digestibilities of individual amino acids.
Tests conducted using five compound diets indicated that ingredient digestibility was additive for protein, amino acids, lipids and energy, whereas the digestibility of carbohydrates in the compound feeds was slightly lower than predicted.
Diets for Sparus aurata may thus be formulated on the basis of digestibility of individual ingredients.     

Food choice and reproductive success of Folsomia candida feeding on copper-contaminated mycelium of the soil fungus Alternaria alternata

We examined collembolan food preference for fungal mycelium grown on copper-contaminated medium, and the relationship between copper content, food selectivity and collembolan fitness when fed contaminated mycelium.To clarify whether collembolan food selectivity is related to fitness parameters, Folsomia candida were fed mycelium of the dark-pigmented fungus Alternaria alternata grown on medium with different copper concentrations. Copper-contaminated food (fungus grown on 50, 125, 250 and 500 μg Cu g^?1 medium, fresh wt.) was offered together with untreated food for 4 weeks. F. candida fed selectively on the provided mycelium and discriminated clearly between mycelium grown on high and low levels of contamination, distinctly preferring fungus grown on medium with a total copper concentration of 50 and 125 μg g^?1. In contrast, fungus grown on highly contaminated medium (250 and 500 μg g^?1) was avoided. Collembolan food preference generally matched fitness parameters. Reproduction was significantly affected by the total copper concentration of the fungal growth medium. When fed their preferred mycelium, collembolan reproduction was enhanced, whereas a diet of highly contaminated mycelium (250 or 500 μg g^?1) resulted in a strong decrease in reproduction. Adult survival was affected only marginally. Even though heavy metal contamination is a potential stress factor for many soil microarthropods, F. candida is able to discriminate between high and low quality food sources, and even benefits from moderately elevated copper concentrations.