Detection of Coxiella burnetii in buffaloes aborted fetuses by IS111 DNA amplification: A preliminary report

The aim of this study was to evaluate by PCR analyses the presence of Coxiella burnetii infection in fetuses of water buffalo (Bubalus bubalis) in the Campania region (Southern Italy). Samples were collected only from aborted fetuses and C. burnetii presence was evaluated by one-tube nested PCR amplification of the IS111 repetitive element. Of the 164 fetuses examined 14 (17.5%) were positive after DNA amplification, showing that C. burnetii occurs in this population of water buffaloes.However, more extensive prevalence studies need to be carried out to define the role of buffaloes as reservoirs for this pathogen and also the role of C. burnetii as an abortive agent in this animal.     

Molecular update on cystic echinococcosis in cattle and water buffaloes of southern Italy

Cystic echinococcosis (CE)--caused by the larval stage (hydatid cyst) of the cestode Echinococcus granulosus--is one of the most widespread zoonoses of veterinary and medical importance. Molecular techniques have allowed the identification of 10 different genotypes (G1-G10) of the parasite. The present paper is an update regarding the E. granulosus genotypes infecting water buffaloes and cattle bred in the Campania region of southern Italy. The molecular study was performed on 30 hydatid cysts (11 from water buffaloes and 19 from cattle). Two different mitochondrial DNA genes, namely the cytochrome c oxidase subunits 1 and the 12S ribosomal DNA (12S rDNA) were used as genetic markers. Three different genotypes of E. granulosus were unequivocally identified, i.e. the G1 (common sheep), G2 (Tasmanian sheep) and G3 (buffalo) genotypes, as well as some G1 and G2 variants. It should be noted that the present study demonstrated for the first time: (i) the presence of the G2 genotype in water buffaloes from a Mediterranean area; and (ii) the fact that the analysed portion of the 12S rDNA gene can not discriminate between the G2 and G3 genotypes of E. granulosus. The finding of the G1, G2 and G3 genotypes in large ruminants from southern Italy is of epidemiological relevance and immediate public health importance because of their recognized infectivity in humans.     

A new procedure to measure the antioxidant activity of insoluble food components

The measurement of antioxidant activity was limited to soluble components to date. Functional groups, which are bound to insoluble matters, may exert antioxidant activity by a surface reaction phenomenon. This hypothesis was tested on the insoluble matters of foods, food ingredients, and Maillard reaction products (MRPs). Insoluble matters were prepared by consecutive washes with water and methanol followed by a lyophilization of the insoluble residue. The measurement was performed by a new procedure using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazil (DPPH) colored radicals. These insoluble matters always showed antioxidant activity. Alkali hydrolysis reduced up to 90% the antioxidant activity of cereal-based insoluble matters, thus confirming that fiber-bound compounds have a major role in their antioxidant activity. The antioxidant activity of the insoluble MRPs was not significantly affected by processing conditions, but severe treatments increased the ratio between insoluble and soluble matters. The contribution of insoluble matter to total antioxidant activity was limited for fruits and vegetables, but it was relevant for cereal-based foods and increased over 50% for dietary-fiber-rich ingredients.     

Diagnosis of Coxiella burnetii-related abortion in Italian domestic ruminants using single-tube nested PCR

Coxiella burnetii, an obligate intracellular parasite with a worldwide distribution, is the causative agent of acute and chronic Q fever in humans. Although infection is often unapparent in cattle, sheep and goats, there is increasing evidence that C. burnetii infection in these species is associated with abortion and stillbirth. This paper describes the introduction of a single-tube nested PCR protocol for the diagnosis of C. burnetii-related abortion in domestic ruminants in Italy. A total of 514 aborted foetuses from cattle (n = 138) and sheep and goat (n = 376), collected from 301 farms, were analyzed from January 2001 to March 2005. Ninety-seven of 514 (18.9%) animals tested PCR-positive, with 16/138 (11.6%) cattle and 81/376 (21.5%) sheep and goat. Eleven of 102 (10.8%) farms with reproductive disorders in cattle and 37/199 (18.6%) farms with reproductive disorders in sheep and goats were infected with C. burnetii. A greater incidence was observed in three of the seven investigated provinces (p < 0.01), with rates of infected farms of up to 23.8%. Data showed that almost all the C. burnetii-related abortions were recorded between October and April (p < 0.01). These findings suggest that Q fever in humans is largely underestimated in Italy, probably because its occurrence is obscured by flu-like symptoms in acute forms.     

Influence of three types of farm management on the seroprevalence of Q fever as assessed by an indirect immunofluorescence assay.

The influence of three types of farm management on the occurrence of Q fever in cattle was investigated by means of a serological study carried out in Irpinia in southern Italy. Twenty-one herds were permanently housed, 26 were housed in winter and turned out to graze in the spring, and six herds were kept outdoors throughout the year. Blood samples were taken from 1188 cattle and tested by an indirect immunofluorescence assay. The overall seroprevalence of Q fever was 14.4 per cent. The herds which were housed in winter and turned out in the spring had the highest seroprevalence of 19.6 per cent, followed by the permanently housed animals with a seroprevalence of 13.2 per cent and the unhoused cattle had a much lower seroprevalence of 1.9 per cent.     

Characterization of the second internal transcribed spacer of ribosomal DNA of Calicophoron daubneyi from various hosts and locations in southern Italy

Isolates of the rumen fluke Calicophoron daubneyi (Digenea: Paramphistomidae) from various hosts and three locations in southern Italy were characterized genetically. The second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) plus flanking 5.8S and 28S sequence (ITS-2+) was amplified from individual rumen flukes by PCR. PCR-linked restriction fragment length polymorphism (PCR-RFLP) analysis was performed using four different restriction endonucleases, and PCR products were sequenced. The PCR analyses from all the C. daubneyi specimens produced identical fragments, and the PCR-RFLP analyses did not show, with respect to any of the four restriction endonucleases, any differences between the C. daubneyi specimens. The sequence analyses of the ITS-2+ from each of the C. daubneyi specimens showed them all to be 428 bp, and composed of the entire ITS-2 sequence (282 bp) plus the two partial flanking conserved sequences, 5.8S (99 bp) and 28S (47 bp). No intra-specific variation was observed in the nucleotide composition of the ITS-2+ (homology=100%). There was, however, an observable inter-specific variation between the ITS-2+ of C. daubneyi and the ITS-2+ of both Calicophoron calicophorum (homology=97.2 %) and Calicophoronmicrobothrioides (homology=97.4 %), both previously deposited in the GenBank. The finding of the present study shows that, as has already demonstrated for other parasitic helminths, ITS-2 can serve as an effective genetic marker for the molecular identification of paramphistomes, and as a useful tool for developing molecular epidemiological techniques for the study of C. daubneyi transmission patterns and prevalence in definitive and intermediate hosts.     

Cystic echinococcosis in water buffaloes: epidemiological survey and molecular evidence of ovine (G1) and buffalo (G3) strains

A survey of cystic echinococcosis (CE) in the water buffalo (Bubalus bubalis) of the Italian Mediterranean breed was carried out in Campania, a region of southern Italy. In addition, a molecular study was performed on 48 hydatid cysts coming from 48 water buffaloes in order to determine the Echinococcus granulosus strain(s) present in this host. Out of a total of 722 water buffaloes examined for CE, 76 (10.5%) were found infected. The average number of cysts per buffalo was 4.3 (minimum 1, maximum 45). Seventeen buffaloes had hydatid cysts only in the liver (with an average of 5 cysts/liver), 34 only in the lungs (with an average of 1.8 cysts/lungs), and 25 buffaloes had cysts both in the liver and in the lungs. Fertile cysts were found in 10 (13.2%) out of the 76 positive buffaloes. The sequencing of the mitochondrial cytochrome C oxidase subunit 1 (CO1) gene of the 48 hydatid cysts produced sequences of 419 bp for each sample analysed. For 33 samples, alignment of the obtained sequences with those present in GenBank showed a total homology with the common domestic sheep strain G1; for 15 samples, sequences obtained showed 100% homology with buffalo strain G3. The findings of the present survey represent the first epidemiological and molecular comprehensive studies on CE in water buffalo from an endemic area for E. granulosus.