Ewe breed differences in cervical anatomy and cervicovaginal mucus properties: An international study.

In sheep, cervical artificial insemination (AI) involves depositing semen at the cervical opening, as it is not possible to traverse the cervix due to its complex anatomy. However, internationally this method yields low pregnancy rates when frozen-thawed semen is used. An exception to this is in Norway, in which vaginal deposition of frozen-thawed semen to a natural estrus yields pregnancy rates around 70%. As the cervix and its secretions are the principal factors influencing sperm transport to the site of fertilization the aim of this study was to characterise the differences in the cervical anatomy as well as the cervicovaginal mucus properties of six European ewe breeds across three countries known to have differences in pregnancy rates following cervical AI with frozen-thawed semen. These were Suffolk and Belclare in Ireland, Fur and Norwegian White Sheep (NWS) in Norway and Ile de France and Romanov in France (n = 28-30 ewes/breed). Cervicovaginal mucus was collected at the follicular and luteal phases of both a synchronized and natural cycle and assessed for mucus weight, viscosity and colour. The anatomical characteristics of the cervix (length of the cervix, number of cervical rings and the appearance of the external os) were assessed post-mortem. There was a type of the cycle by ewe breed interaction represented by no differences in mucus production between ewe breeds at the natural cycle for both the follicular and luteal phases of the cycle. However, there were differences between ewe breeds at the synchronized cycle (P < 0.05). Belclare had the lowest mucus production at the follicular phase while NWS had the lowest amount of mucus at the luteal phase of the synchronized cycle. Overall, across all ewe breeds, mucus production was higher at the follicular than at the luteal phase (P < 0.05). Despite reports of Suffolk and NWS having the most divergent pregnancy rates following cervical AI with frozen-thawed semen, both breeds had the lowest overall mucus viscosity at the follicular phase of both types of cycle with no differences between both ewe breeds (P > 0.05). The length of the cervix, number of cervical rings and the external os type were affected by ewe breed (P < 0.05). Suffolk ewes had longer cervices but lower number of cervical rings than NWS and Fur ewes (both with higher pregnancy rates). In conclusion, while mucus production and mucus viscosity was affected by breed, these changes are not consistent with the known differences between ewe breeds in their pregnancy rates following cervical AI with frozen-thawed semen.

The distribution limit of the common tick, Ixodes ricinus, and some associated pathogens in north-western Europe.

In north-western Europe, the common tick, Ixodes ricinus, is widely established, its distribution appears to be increasing and the spread of tick-borne diseases is of increasing concern. The project 'Flått i Nord' (Ticks in northern Norway) commenced in spring 2009 with the intention of studying the tick's distribution and that of its pathogens in northern Norway. Several methods were used: cloth-dragging, collecting from trapped small mammals, and collecting from pets. Since 2010, the occurrence of ticks in the region of northern Norway was determined directly by cloth-dragging 167 times in 109 separate locations between the latitudes of 64 °N and 70 °N (included seven locations in the northern part of Trøndelag County). The northernmost location of a permanent I. ricinus population was found to be Nordøyvågen (66.2204 °N, 12.59 °E) on the Island of Dønna. In a sample of 518 nymphal and adult ticks, the Borrelia prevalence collected close to this distribution limit varied but was low (1-15 %) compared with the locations in Trøndelag, south of the study area (15-27 %). Five specimens (1 %) were positive for Rickettsia helvetica. The length of the vegetation growing season (GSL) can be used as an approximate index for the presence of established populations of I. ricinus. The present study suggests that the threshold GSL for tick establishment is about 170 days, because the median GSL from 1991 to 2015 was 174-184 days at sites with permanent tick populations, showing a clear increase compared with the period 1961-1990. This apparent manifestation of climate change could explain the northward extension of the range of I. ricinus.

Neonatal Anaplasma platys infection in puppies: Further evidence for possible vertical transmission.

Anaplasma platys, the aetiological agent of infectious canine cyclic thrombocytopaenia, infects platelets of dogs, usually causing mild or asymptomatic disease. Although A. platys is transmitted by ticks, as for other Anaplasma species, alternative modes of transmission may be involved. The aim of the present study was to evaluate the occurrence of A. platys infection in litters of puppies, which could suggest possible vertical transmission. Twelve litters, together with the respective bitches, were included in the study for the detection of A. platys DNA by PCR, followed by sequencing. Five puppies, from 2/4 litters <28 days of age, tested positive for A. platys DNA. No puppies from eight litters 1-3 months of age tested positive for A. platys DNA. The identical sequences (16S rRNA and gltA partial gene), the absence of ticks on puppies at the time of collection and the young age of the five infected puppies suggest vertical transmission of A. platys. This mode of transmission might contribute to the maintenance and spread of the pathogen in canine populations.

Temporal and spatial variation in Anaplasma phagocytophilum infection in Swedish moose (Alces alces).

SUMMARY: The occurrence of Anaplasma phagocytophilum was investigated in spleen and serum samples from Swedish moose (Alces alces) in southern Sweden (island and mainland). Samples were analysed for presence of A. phagocytophilum DNA by real-time PCR (n = 263), and for Anaplasma antibodies with ELISA serology (n = 234). All serum samples had antibodies against A. phagocytophilum. The mean DNA-based prevalence was 26·3%, and significant (P < 0·01) temporal, and spatial variation was found. Island moose had significantly (P < 0·001) higher prevalence of A. phagocytophilum DNA than moose from the mainland areas. Two samples were sequenced to determine genetic variation in the 16S rRNA and groESL genes. Genetic sequence similarity with the human granulocytic anaplasmosis agent, equine granulocytic ehrlichiosis agent, and different wildlife-associated A. phagocytophilum variants were observed in the 16S rRNA and groESL genes. Our study shows that moose are exposed to A. phagocytophilum in Sweden, and represent a potential wildlife reservoir of the pathogen.

Anaplasma phagocytophilum - the most widespread tick-borne infection in animals in Europe.

The bacterium Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) may cause infection in several animal species including human. The disease in domestic ruminants is also called tick-borne fever (TBF), and has been known for at least 200 years. In Europe, clinical manifestations due to A. phagocytophilum have been recorded in sheep, goat, cattle, horse, dog, cat, roe deer, reindeer and human. However, seropositive and PCR-positive mammalian have been detected in several other species. Investigations indicate that the infection is prevalent in Ixodes ricinus areas in most countries in Europe. A. phagocytophilum infection may cause high fever, cytoplasmatic inclusions in phagocytes and severe neutropenia, but is seldom fatal unless complicated by other infections. Complications may include abortions, and impaired spermatogenesis for several months. However, the most important aspect of the infection at least in sheep is its implication as a predisposing factor for other infections. Factors such as climate, management, other infections, individual conditions etc. are important for the outcome of the infection. A. phagocytophilum may cause persistent infection in several species. Based on the 16S rRNA gene sequences several variants exist. Different variants may exist within the same herd and even simultaneously in the same animal. Variants may behave differently and interact in the mammalian host.

Characterization of Anaplasma phagocytophilum major surface protein 5 and the extent of its cross-reactivity with A. marginale.

Major surface protein 5 (Msp5) of Anaplasma marginale is highly conserved in the genus Anaplasma and the antigen used in a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for serologic identification of cattle with anaplasmosis. This study analyzes the degrees of conservation of Msp5 among various isolates of Anaplasma phagocytophilum and the extent of serologic cross-reactivity between recombinant Msp5 (rMsp5) of Anaplasma marginale and A. phagocytophilum. The msp5 genes from various isolates of A. phagocytophilum were sequenced and compared. rMsp5 proteins of A. phagocytophilum and A. marginale were used separately in an indirect ELISA to detect cross-reactivity in serum samples from humans and dogs infected with A. phagocytophilum and cattle infected with A. marginale. Serum samples were also tested with a commercially available competitive ELISA that uses monoclonal antibody ANAF16C1. There were 100% sequence identities in the msp5 genes among all of the A. phagocytophilum isolates from the United States and a horse isolate from Sweden. Sheep isolates from Norway and dog isolates from Sweden were 99% identical to one another but differed in 17 base pairs from the United States isolates and the horse isolate. Serologic cross-reactivity was identified when serum samples from cattle infected with A. marginale were reacted with rMsp5 of A. phagocytophilum and when serum samples from humans and dogs infected with A. phagocytophilum were reacted with rMsp5 of A. marginale in an indirect-ELISA format. Serum samples from dogs or humans infected with A. phagocytophilum did not cross-react with rMsp5 of A. marginale when tested with the commercially available cELISA. These results suggest that rMsp5 of A. phagocytophilum is highly conserved among United States and European isolates and that serologic distinction between A. phagocytophilum and A. marginale infections cannot be accomplished if rMsp5 from either organism is used in an indirect ELISA.

Detection by the polymerase chain reaction of Anaplasma phagocytophilum in tissues of persistently infected sheep.

To investigate the reservoir tissues of the tick-borne bacterium Anaplasma phagocytophilum in persistently infected sheep, six 6-month-old lambs were infected with a field isolate of the bacterium and maintained under tick-free conditions. At one and two weeks post-infection, A. phagocytophilum was detected in the peripheral blood of all lambs by examining May-Grünwald Giemsa-stained blood smears for classical intra-neutrophil inclusions, and by an A. phagocytophilum-specific nested PCR. After euthanasia at 3 months post-inoculation, peripheral blood and numerous tissue samples were collected from each lamb. DNA extracted from these samples was then subjected to PCR. All blood samples were PCR-negative but three lambs had PCR-positive tissues including intestinal wall and lymph nodes, thymus, bone marrow, kidney and bladder wall. The widespread nature of PCR-positive tissues suggested that circulatory cells may form the reservoir cells for A. phagocytophilum infection in carrier sheep, rather than lymphoid tissues as in rodents. PCR-positive tissue and blood samples were strikingly fewer in the experimentally infected sheep than reported earlier in tick-exposed carrier sheep under field conditions. It seems possible that tick infestation amplifies A. phagocytophilum infections in carrier sheep to a degree that enables tick transmission to occur.