39This study explored various aspects of the epidemiology of paramphistomosis in

BackgroundRumen flukes are trematode parasites found globally; in tropical and sub-tropical climates, infection can result in paramphistomosis, which can have a deleterious impact on livestock. In Europe, rumen fluke is not regarded as a clinically significant parasite, recently however, the prevalence of rumen fluke has sharply increased and several outbreaks of clinical paramphistomosis have been reported. Gaining a better understanding of rumen fluke transmission and identification of risk factors is crucial to improve the control of this parasitic disease. In this regard, a national prevalence study of rumen fluke infection and an investigation of associated risk factors were conducted in Irish sheep flocks between November 2014 and January 2015. In addition, a molecular identification of the rumen fluke species present in Ireland was carried out using an isolation method of individual eggs from faecal material coupled with a PCR. After the DNA extraction of 54 individual eggs, the nuclear fragment ITS-2 was amplified and sequenced using the same primers.ResultsAn apparent herd prevalence of 77.3 % was determined. Several risk factors were identified including type of pasture grazed, regional variation, and sharing of the paddocks with other livestock species. A novel relationship between the Suffolk breed and higher FEC was reported for the first time. The predominant rumen fluke species found was C. daubneyi. Nevertheless, P. leydeni was unexpectedly identified infecting sheep in Ireland for the first time.ConclusionsAn exceptionally high prevalence of rumen fluke among Irish sheep flocks has been highlighted in this study and a more thorough investigation is necessary to analyse its economic impact. The isolation of individual eggs coupled with the PCR technique used here has proven a reliable tool for discrimination of Paramphistomum spp. This technique may facilitate forthcoming studies of the effects of paramphistomosis on livestock production. The most noteworthy finding was the identification of P. leydeni affecting sheep in Ireland, however further studies are required to clarify its implications. Also, a significant relationship between Suffolk breed and a heavier infection was found, which can be used as a starting point for future research on control strategies of rumen fluke infection.

The prevalence and aetiology of natural paramphistomosis was investigated in cattle slaughtered in the Castilla y León region (Spain) over a 3 year-period. The overall prevalence of positive animals was 6.20%. The parasite burden per animal ranged from 8 to 8005 (median=144) and the ruminal atrium had the highest parasite burden whereas the ruminal dorsal sac the lowest. The prevalence and parasite burden increased with age while these parameters were lower in cattle under intensive management. Calicophoron daubneyi was the only Paramphistomidae species identified using morphoanatomical, histological and molecular methods in the studied animals.

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.