Six-year longitudinal study of Fasciola hepatica bulk milk antibody ELISA in the dairy dense region of the Republic Ireland.

Completion of the F. hepatica lifecycle is dependent on suitable climatic conditions for development of immature stages of the parasite, and its snail intermediate host. Few investigations have been conducted regarding temporal variations in F. hepatica status in Irish dairy herds. The current study aimed to conduct a longitudinal study examining annual and seasonal trends in bulk milk seropositivity over six years, while also investigating associations with soil temperature, rainfall and flukicide treatment. Monthly bulk milk samples (BTM) were submitted by 28 herds between March 2009 and December 2014. In all, 1337 samples were analysed using a Cathepsin L1 ELISA. Soil temperature, rainfall and management data were obtained for general estimating equation and regression analyses. A general decrease in milk seropositivity was observed over the six year study period and was associated with an increased likelihood of treating for liver fluke (OR range=2.73-6.96). Annual and seasonal analyses of rainfall and F. hepatica BTM status yielded conflicting results. Higher annual rainfall (>1150mm) yielded a lower likelihood of being BTM positive than annual rainfall of <1000mm (OR=0.47; P=0.036). This was most likely due to farmers being more proactive in treating for F. hepatica in wetter years, although a 'wash effect' by high rainfall of the free living stages and snails cannot be ruled out. Higher seasonal rainfall (>120mm), however, was associated with increased ELISA S/P% values (Coefficient=9.63S/P%; P=0.001). Soil temperature was not found to influence F. hepatica to the same extent as rainfall and may reflect the lack of severe temperature fluctuations in Ireland. Flukicides active against both immature and mature F. hepatica were approximately half as likely to record a positive F. hepatica herd BTM status than a flukicide active against only the mature stage of the parasite (OR≅0.45; P<0.01). This study highlights the importance of examining both annual and seasonal F. hepatica data, which can vary significantly. Additionally, it highlights the progress that can be achieved in fluke control by application of a continuous BTM monitoring program.

Prevalence of BoHV-1 seropositive and BVD virus positive bulls on Irish dairy farms and associations between bull purchase and herd status.

BACKGROUND: BVD and IBR are contagious viral diseases highly prevalent in Irish cattle. Despite their significant reproductive and economic impact very little is known about the BVD and IBR status of stock bulls (a bull used for breeding purposes). There are still a high proportion of dairy farms in Ireland that rely on the use of a bull for breeding cattle and ensuring the fertility of the bulls is of paramount importance for the efficiency of the farms. The prevalence of BoHV-1 and BVD in stock bulls in Irish dairy herds has never been investigated. The objectives of this study therefore were: (i) to provide descriptive, observational data on the use of stock bulls on Irish dairy farms; (ii) to investigate the BVD and BoHV1 status of a sub-set of stock bulls; (iii) to investigate factors associated with BVD and BoHV1 status of stock bulls and (iv) to investigate factors associated with dairy herd status for BVD and BoHV1, including any associations with the use of stock bull. A total of 529 blood samples from bulls involved in the dairy breeding process were analysed for BVD virus using RT-PCR, and BoHV-1 antibodies by ELISA test. A total of 305 different dairy herds took part in the study and the overall BVD and BoHV-1 herd status was determined by ELISA using four bulk tank milk samples over the 2009 lactation. Logistic regression was used to investigate the associations between the stock bulls and BVD and BoHV-1 herd and individual status. RESULTS: Of the 305 total participating farms, 235 farms (77 %) had at least one bull and 167 farms had purchased bulls. Two bulls (0.4 %) out of 529 tested were found positive for BVD virus and 87 (16.7 %) tested seropositive for BoHV-1. Some significant associations were identified between the purchase of bulls and both viral diseases. Purchased bulls were three times more likely to be seropositive for BoHV-1 than homebred bulls. In the same way, herds with purchased bulls were three times more likely to be classified as seropositive for BVD and four times more likely to have evidence of recent BoHV-1 circulation than farms where all the bulls were homebred. CONCLUSIONS: The prevalence of BoHV-1 and BVD in stock bulls in Irish dairy herds has never been investigated. This study highlights the widespread use of stock bulls in Irish dairy herds, as well as the high rate of exchange of bulls between farms. Significant associations were found between the origin of the bull and their serological BoHV-1 status. In keeping with these results, bulls with higher number movements between farms were more likely to be seropositive for BoHV-1.

Analysis of an expressed sequence tag library from Dicrocoelium dentriticum.

Dicrocoeliosis caused by Dicrocoelium dendriticum is an important liver disease, which affects ruminants all around the world. Despite the significant economic losses caused by this trematode, molecular knowledge is very scarce. In fact, there is no information in the expressed sequence tag (EST) database about the parasite. Furthermore, the immunological diagnosis of dicrocoeliosis remains unsatisfactory, and there aren't available recombinant proteins that could be tested in the diagnosis. For this reason a cDNA library was constructed with mRNA extracted from D. dendriticum adults for first time. A random preliminary screening of 230 phage plaques from the library resulted in the identification of 173 new EST. The deduced proteins expressed by these genes have been described as possible vaccine targets in other trematodes, and/or as relevant diagnosis antigens. Then, our goal was to identify D. dentriticum diagnosis genes to be used as recombinant antigens in the specific immunological diagnosis of the trematodoses. A D. dendriticum cDNA encoding an 8-kDa recombinant protein has been cloned, expressed in Escherichia coli and evaluated in dicrocoeliosis diagnosis using both Western Blot and enzyme-linked immunosorbent assay (ELISA). The recombinant expression molecule has demonstrated its value as a diagnosis antigen of dicrocoeliosis, able to discriminate between positive and controls on day 30 post infection. This is the first research conducted for identification and characterization of D. dendriticum ESTs, which can serve as a starting point for future research on immunodiagnosis and immunoprofilaxis of dicrocoeliosis.

Development and validation of a mtDNA multiplex PCR for identification and discrimination of Calicophoron daubneyi and Fasciola hepatica in the Galba truncatula snail.

Paramphistomosis and Fasciolosis caused by Calicophoron daubneyi and Fasciola hepatica, respectively, are frequent and important trematodoses in ruminant livestock worldwide. Both parasites use the same snail, Galba truncatula, as intermediate host. The aim of this study was to develop and validate an analytical method based on a mitochondrial DNA (mtDNA) multiplex PCR technique which would allow the early and specific identification, in one step, of C. daubneyi and F. hepatica infection in G. truncatula. First of all, a 1035 bp fragment of mtDNA from adult C. daubneyi worms was obtained. Then two pairs of specific mtDNA primers, which amplified a DNA fragment of 885 pb in the case of C. daubneyi, and of 425 pb in that of F. hepatica, were designed. By means of the multiplex PCR technique developed, there was always a specific amplification in samples from adult F. hepatica and C. daubneyi, but not from Calicophoron calicophorum, Cotylophoron cotylophorum, Cotylophoron batycotyle or Dicrocoelium dendriticum. Likewise, specific amplifications of the expected DNA fragments happened in all samples from snails harbouring larval stages of C. daubneyi or F. hepatica, previously detected by microscopy. However, amplifications were not seen when DNA from snails harbouring other Digenea (Plagiorchiidae, Notocotylidae and furcocercous cercariae) was analysed. Moreover, DNA from G. truncatula molluscs free from infection was not amplified. The multiplex PCR assay permitted infection in the snails experimentally infected with 4 miracidia to be detected as early as day 1 p.i. in the case of F. hepatica and with only 2 miracidia from day 2 p.i. in both, C. daubneyi and F. hepatica. Nevertheless it was necessary to wait until days 29 and 33 p.i. to see C. daubneyi and F. hepatica immature redia, respectively, using microscope techniques. The detection limit of the PCR technique was very low: 0.1 ng of DNA from C. daubneyi and 0.001 ng of DNA from F. hepatica. This allowed infection by either F. hepatica or C. daubneyi to be detected even when pools made up with only 1 µl (60 ng of DNA) from infected snail plus 99 µl from non-infected ones were analyzed. Moreover, simultaneous detection of both parasites was experimentally possible in pools made up with uninfected (98 µl), C. daubneyi infected (1 µl) and F. hepatica infected (1 µl) snails. The most precise and early diagnosis of the infections using the multiplex PCR technique designed will allow more realistic epidemiological models of both infections to be established and consequently a better strategic control.

Proteomic analysis of the tegument and excretory-secretory products of Dicrocoelium dendriticum (Digenea) adult worms.

Dicrocoeliosis, caused by Dicrocoelium dendriticum, is an important hepatic parasitosis in ruminants, whose immunological diagnosis and control remain unsatisfactory. There are very few studies on the antigens of this trematode and molecular knowledge about it is practically nil. Therefore the aim of this study was to identify the major antigenic proteins in the tegument (TG) and excretory-secretory (ES) antigenic extracts of D. dendriticum. The separation conditions of the protein extracts were optimized using 2-D PAGE; the gels were stained with colloidal Coomassie or transferred to carry out immunodetection with anti-Dicrocoelium dendriticum sera. The proteins of interest excised from the gels were identified by mass spectrometry (MALDI). The proteomic maps of the TG and ES extracts of D. dendriticum were defined first, detecting 332 spots in the TG and 284 in the ES, with a similar distribution in both. A quantity of 29 proteins in the excretion-secretion products and 43 in the teguments were identified first in D. dendriticum, 23 of them antigenic, involved in various processes such as: metabolism, detoxification, chaperone, transport or structural molecules. These results could help us to understand the complex parasite-host relationships, improve the diagnosis of dicroceliosis and help to produce possible vaccines to control it.

Detection of Dicrocoelium dendriticum larval stages in mollusc and ant intermediate hosts by PCR, using mitochondrial and ribosomal internal transcribed spacer (ITS-2) sequences.

The aim of this study was to develop, perfect and validate the PCR (polymerase chain reaction) technique using mitochondrial (mt) and ribosomal (ITS-2) DNA for the accurate identification of Dicrocoelium dendriticum in molluscs and ants, the first and second intermediate hosts, and their early detection. The first primers that were designed amplified a 169 pb mtDNA fragment of D. dendriticum permitted the detection of a single D. dendriticum metacercaria from the Formica rufibarbis and Formica pratensis abdomen, as well as the detection of the brainworm in the head of the ants collected in tetania. Although these primers did not amplify Dicrocoelium chinensis DNA and permitted detected D. dendriticum in the molluscs, they did not discriminate Brachylaimidae metacercariae found in the same mollusc. The PCR that was designed to amplify a 93 bp fragment of the ITS-2 is D. dendriticum specific as it did not amplify D. chinensis, Brachylaimidae and other trematodes. This technique is very sensitive since it permitted the detection of D. dendriticum in the molluscs from the first day post-infection, the brainworm in the head of the ants and only 1 D. dendriticum metacercaria from the abdomen of the ants. Both techniques are important, mainly the latter.