Anaplasma phagocytophilum up-regulates some anti-apoptotic genes in neutrophils and pro-inflammatory genes in mononuclear cells of sheep.

Anaplasma phagocytophilum, the causative agent of tick-borne fever (TBF) in sheep and cattle and human granulocytic anaplasmosis, has the unique ability to selectively infect and multiply within the hostile environment of the neutrophil. Previous studies have shown that sheep with TBF are more susceptible to other infections and that infected neutrophils have reduced phagocytic ability and delayed apoptosis. This suggests that survival of A. phagocytophilum in these short-lived cells involves the ability to subvert or resist their bacterial killing, but also to modify the host cells such that the host cells survive long after infection. The present study shows that infection of sheep by A. phagocytophilum is characterized by up-regulation of some anti-apoptotic genes (BCL2, BIRC3 and CFLAR) in neutrophils and up-regulation of genes encoding the pro-inflammatory cytokines interferon-γ, interleukin (IL)-1ß and IL-6 in mononuclear cells during the period of bacteraemia. Infection with A. phagocytophilum was also characterized by significant up-regulation of CYBB, which is associated with the respiratory burst of neutrophils.

Recurrent bacteraemia in sheep infected persistently with Anaplasma phagocytophilum.

Following experimental or natural infection with Anaplasma phagocytophilum, the causative agent of tick-borne fever (TBF), sheep may be infected persistently for several months or years. In the present study, quantitative real-time polymerase chain reaction was used to investigate the duration and magnitude of primary bacteraemia and to establish whether the organism is present continuously in the peripheral blood after the period of primary bacteraemia and the cessation of clinical signs. Persistent infection was characterized by a clearly defined period of primary bacteraemia followed by recurrent cycles of bacteraemia, usually lasting a few days and of lower magnitude, interspersed by negative periods of variable duration in which bacterial DNA could not be detected. During a 150-day period of consecutive sampling of four sheep, A. phagocytophilum was detected on 64.25 ± 4.9 occasions, which means that on average bacterial DNA was detected in 42.8 ± 3.3 percent of all samples, with the positive days falling into 15-20 distinct cycles. Primary bacteraemia lasted for 15.5 ± 2.33 days, but secondary and subsequent cycles of bacteraemia were short-lived, with 61% of the cycles lasting only 1-2 days and 39% lasting for 3 or more days. Secondary and subsequent cycles of bacteraemia were not accompanied by febrile responses or other clinical features of TBF. For three animals, bacterial DNA was detected at 311, 318 and 358 days post infection, indicating the long-term persistence of A. phagocytophilum within peripheral blood.

California mastitis test scores as indicators of subclinical intra-mammary infections at the end of lactation in dairy cows.

Intramammary infections (IMI) during the dry period can be reduced through the use of dry cow therapy (DCT); in the future, its blanket use is likely to be questioned in the light of public concern regarding the routine use of antibiotics in food producing animals. One possible alternative is to limit DCT to cows with IMI just before drying off, which would require a quick, simple identification of sub-clinical IMI. In the present study we examined quarter milk samples obtained from 240 cows one week before and on the day of drying off, using the California mastitis test (CMT) and for IMI by bacteriological culture. The results indicated that high CMT scores at drying off may be good indicators of IMI: there was a significant association between the frequency of isolation of major pathogens and the CMT score in milk samples obtained one week before (Pearson's χ(2)=27.04, df=4, p<0.001) and those at drying off (Pearson's χ(2)=25.87, df=4, p<0.001).

Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Anaplasma phagocytophilum in sheep.

An indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Anaplasma phagocytophilum in ovine serum samples was evaluated. The assay used purified A. phagocytophilum grown in tick cell cultures as antigen. Serum samples were diluted 1 in 200 and binding was detected with anti-sheep IgG conjugated to horseradish peroxidase. All tests were carried out in the presence of positive and negative control samples. Optical density (OD) values obtained for each test sample at 490 nm were used to calculate percentage positivity (PP) of each sample based on the ratio of the OD of the test sample that of the positive reference sample. Known negative samples (n=69) obtained from uninfected sheep bred and maintained in a tick-free environment and subsequently shown to be susceptible to A. phagocytophilum were used to establish the cut-off point between negative and positive samples and to establish the specificity of the test. Serum samples obtained from 92 animals 14-21 days after infection were used to establish the sensitivity of the test. Using a cut-off point of 20PP (mean+2 standard deviations of the PP of 69 control samples) the test was shown to have a sensitivity of 84.8% and a specificity of 95.7%. Lowering the cut-off point to 15PP increased the sensitivity to 94.6%, but reduced the specificity to 92.8%.

The effect of dry cow therapy and internal teat-sealant on intra-mammary infections during subsequent lactation.

Dry cow therapy (DCT), an infusion of antibiotics into the mammary gland at the end of lactation, is widely used for the control of intra-mammary infections (IMI) in the dairy cow. However, increased public health concerns about the use of antibiotics, has led to the search for alternatives to the routine use of antibiotics during drying off. In the present study the effects of three dry cow treatments, two types of DCT and a teat-sealant, on the development of new IMI and clinical mastitis were investigated in 240 cows belonging to two herds (Herd A and Herd B). In Herd A, 60 cows were given one type of DCT (Cloxacillin) and the other 60 cows were given another type of DCT (Framycetin). In Herd B, 60 cows were given teat-sealant and the other herd were not given any treatment. There were significantly more new IMI at calving in control cattle compared to those given teat-sealants (p<0.001) and there were more cases of clinical cases of mastitis in the control group. The number of clinical cases detected in cows given teat-sealant in Herd B were not significantly different from those detected in cows in Herd A given Cloxacillin or Framycetin.

In-vitro studies on the susceptibility of ovine strains of Anaplasma phagocytophilum to antimicrobial agents and to immune serum.

Five strains of Anaplasma phagocytophilum, the causative agent of tick-borne fever of sheep and cattle and human granulocytic anaplasmosis, were tested in vitro for their susceptibility to nine antibiotics using the continuous tick cell line ISE6. Minimum inhibitory concentrations (MICs) were evaluated by comparing the percentage of infection of Giemsa-stained antibiotic-treated Anaplasma phagocytophilum-infected cells with that of untreated controls after 6 days of culture. The minimum bactericidal concentrations (MBCs) were evaluated after washing infected cells with antibiotic-free medium and further incubation of 6 days before comparing the percentage of infection of Giemsa-stained antibiotic-treated and untreated cells and by comparing the number of copies of the p44 gene by real-time polymerase chain reaction using p44-specific primers. The tick cell culture system was also used to assay the possible neutralizing effects of immune serum on cell-free bacteria in vitro. The neutralizing effects of immune serum were evaluated by comparing the number of copies of the p44 gene in samples inoculated with cell-free bacteria after 1h incubation with two-fold dilutions of immune sera obtained 21 days after infection with those inoculated with cell-free bacteria after incubation for 1h with two-fold dilutions of sera obtained before infection. Doxycycline, rifampin and ciprofloxacin were the most effective compounds, with MICs of 0.125 microg/ml, 0.5 mg/ml and 1 microg/ml, respectively. There was total resistance to penicillin, ampicillin, ceftriaxone and streptomycin and only very limited susceptibility to gentamycin and chloramphenicol. Both doxycycline and rifampin were also bactericidal at the same concentrations. Exposure of bacteria to immune ovine sera resulted in significant reductions of the number of copies of p44 gene.

Epidemiology of bovine venereal campylobacteriosis: geographic distribution and recent advances in molecular diagnostic techniques.

Bovine venereal campylobacteriosis (BVC) is a major cause of economic loss to the cattle industries in different parts of the world. Camplylobacter fetus subsp. venerealis (Cfv), the main causative agent of BVC, is highly adapted to the genital tract of cattle and is transmitted by carrier bulls. However, infertility and abortions can also be caused by the intestinal pathogens C. fetus subsp. fetus (Cff), and C. jenuni, which are not venereally transmitted. Bovine venereal campylobacteriosis, caused by Cfv associated with lowered fertility, embryo mortality and abortion, repeated returns to service, reduced pregnancy rates and extended calving intervals, has the highest prevalence in developing countries where natural breeding in cattle is widely practised. The epidemiology, pathogenesis and diagnosis of the disease have been the subject of previous reviews. The main focus of this review is to highlight the epidemiology of this disease with particular reference to geographical distribution and recent advances in molecular diagnostic techniques. It is hoped that further research interest of scientists will be stimulated with a view to finding lasting solutions to the reproductive problems associated with the disease for better livestock productivity, particularly in developing endemic countries.

Diazinon oxon interferes with differentiation of rat C6 glioma cells.

The purpose of this study was to evaluate the toxicity of diazinon oxon (DZO), a major in vivo metabolite of the organophosphate insecticide diazinon (DZ), on differentiating rat C6 glioma cells. At concentrations shown to be non-cytotoxic by both the MTT and the Kenacid blue dye binding assays (1, 5 and 10 microM), DZO caused after 24h a reduction in the number of extensions developed from C6 cells induced to differentiate by serum withdrawal and addition of sodium butyrate. Densitometric scanning of Western blots of extracts of C6 cells demonstrated that, at all concentrations used, DZO decreased after 24h the expression of glial fibrillary acidic protein (GFAP) compared to controls. In addition, exposure to 10 microM DZO for 24h reduced the levels of tubulin and microtubule associated protein 1B (MAP1B). On the other hand, levels of MAP2c were not affected by DZO treatment. In contrast to our previous data on DZ, the above findings suggest that its oxon metabolite, DZO, may, at biologically relevant, subcytotoxic concentrations, interfere with glial cell differentiation.

Relative importance of Ixodes ricinus and Ixodes trianguliceps as vectors for Anaplasma phagocytophilum and Babesia microti in field vole (Microtus agrestis) populations.

The importance of Ixodes ricinus in the transmission of tick-borne pathogens is well recognized in the United Kingdom and across Europe. However, the role of coexisting Ixodes species, such as the widely distributed species Ixodes trianguliceps, as alternative vectors for these pathogens has received little attention. This study aimed to assess the relative importance of I. ricinus and I. trianguliceps in the transmission of Anaplasma phagocytophilum and Babesia microti among United Kingdom field voles (Microtus agrestis), which serve as reservoir hosts for both pathogens. While all instars of I. trianguliceps feed exclusively on small mammals, I. ricinus adults feed primarily on larger hosts such as deer. The abundance of both tick species and pathogen infection prevalence in field voles were monitored at sites surrounded with fencing that excluded deer and at sites where deer were free to roam. As expected, fencing significantly reduced the larval burden of I. ricinus on field voles and the abundance of questing nymphs, but the larval burden of I. trianguliceps was not significantly affected. The prevalence of A. phagocytophilum and B. microti infections was not significantly affected by the presence of fencing, suggesting that I. trianguliceps is their principal vector. The prevalence of nymphal and adult ticks on field voles was also unaffected, indicating that relatively few non-larval I. ricinus ticks feed upon field voles. This study provides compelling evidence for the importance of I. trianguliceps in maintaining these enzootic tick-borne infections, while highlighting the potential for such infections to escape into alternative hosts via I. ricinus.

The immune status of the bovine uterus during the peripartum period.

The post-partum period in cattle is characterised by an increased risk of infection of the uterus, as the anatomical barriers are broached during parturition and remain open for several days. Infection of the uterus is largely influenced by the balance between bacterial contamination and the local and systemic immune status during pregnancy and around parturition. Infectious diseases are more prevalent during this period, because of an impaired immune status before and immediately after parturition. Neutrophils play a primary role in the defence of the uterus against infection. Influx of neutrophils into the uterus is thought to be mediated by chemoattractants, chemokines and adhesion molecules, such as beta2-integrin (complement receptor 3) and L-selectin (CD62L). Other cellular components activated in the uterus during this period include lymphocytes, eosinophils, mast cells and macrophages. The major classes of immunoglobulins (IgM, IgA and IgG), either by passive diffusion or local production, play an important protective role in the uterus by acting as opsonins to enhance phagocytosis, stimulating the complement pathways or blocking pathogens from adhering to mucosal surfaces. Endometrial cells express toll-like receptor 4 (TLR4), which recognises lipopolysaccharides of Escherichia coli and other Gram negative bacteria, the most common causes of bovine endometritis. Activation of TLR4 triggers the production of tumour necrosis factor alpha and other pro-inflammatory cytokines. The periparturient period is also characterised by an increased secretion of prostaglandin F(2alpha), which enhances uterine immune defences.

Sympatric Ixodes trianguliceps and Ixodes ricinus ticks feeding on field voles (Microtus agrestis): potential for increased risk of Anaplasma phagocytophilum in the United Kingdom?

The importance of wild rodents as reservoirs of zoonotic tick-borne pathogens is considered low in the United Kingdom because, in studies to date, those parasitized by exophilic Ixodes ricinus ticks carry almost exclusively larvae and thus have a minor role in transmission cycles. In a cross-sectional study, 11 (6.7%) of 163 field voles (Microtus agrestis) captured at field sites in Northern England were PCR-positive for Anaplasma phagocytophilum. The voles were found to act as hosts for both larval and nymphal I. ricinus and all stages of the nidicolous tick I. trianguliceps, and eight individuals were infested with ticks of both species at the same time. Two of 158 larval and one of 13 nymphal I. ricinus, as well as one of 14 larval and one of 15 nymphal I. trianguliceps collected from the rodents were PCR-positive. These findings suggest that habitats where field voles are abundant in the United Kingdom may pose a risk of A. phagocytophilum infection because (i) field voles, the most abundant terrestrial mammal in the United Kingdom, may be a competent reservoir; (ii) the field voles are hosts for both nymphal and larval ixodid ticks so they could support endemic cycles of A. phagocytophilum; and (iii) they are hosts for nidicolous I. trianguliceps, which may alone maintain endemic cycles, and exophilic I. ricinus ticks, which could act as a bridge vector and transmit infections to humans and domesticated animals.

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.

Serologic cross-reactivity between Anaplasma marginale and Anaplasma phagocytophilum.

In the context of a serosurvey conducted on the Anaplasma marginale prevalence in Swiss cattle, we suspected that a serological cross-reactivity between A. marginale and A. phagocytophilum might exist. In the present study we demonstrate that cattle, sheep and horses experimentally infected with A. phagocytophilum not only develop antibodies to A. phagocytophilum (detected by immunofluorescent-antibody assay) but also to A. marginale (detected by a competitive enzyme-linked immunosorbent assay). Conversely, calves experimentally infected with A. marginale also developed antibodies to A. phagocytophilum using the same serological tests. The identity of 63% determined in silico within a 209-amino-acid sequence of major surface protein 5 of an isolate of A. marginale and one of A. phagocytophilum supported the observed immunological cross-reactivity. These observations have important consequences for the serotesting of both, A. marginale and A. phagocytophilum infection of several animal species. In view of these new findings, tests that have been considered specific for either infection must be interpreted carefully.

Antigenicity of ovine strains of Anaplasma phagocytophilum grown in tick cells and ovine granulocytes.

Antigens prepared from ovine granulocytes and tick cells infected with ovine strains of Anaplasma phagocytophilum, the causative agent of tick-borne fever, were tested in respect of their suitability for the assay of antibodies in ovine sera by enzyme-linked immunosorbent assay (ELISA) and by indirect immunofluorescent antibody test (IFAT). Antigens prepared from tick cells were as sensitive and specific as those expressed in ovine granulocytes for the detection of specific antibodies by ELISA, but they failed to react in the IFAT with immune sera obtained from sheep previously infected with ovine strains of A. phagocytophilum.

Groupings of highly similar major surface protein (p44)-encoding paralogues: a potential index of genetic diversity amongst isolates of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is a tick-borne bacterium that is zoonotic in the USA and southern Europe, but although the bacterium is endemic in the UK, no cases of clinical human disease have yet been detected in that country. Potential genomic differences amongst UK and USA isolates were investigated by comparing partial 16S rRNA gene and p44 paralogue sequences amplified by PCR from 10 UK ruminant or tick isolates, with published sequences from USA isolates. No significant clustering among the isolates was resolved by phylogenetic analysis of alignments containing 16S rRNA gene sequences. The structure of predicted proteins encoded by p44 paralogues, amplified from 81 clones obtained from the UK isolates, was similar to that described previously for paralogues from USA isolates. Paralogue sequences did not obviously cluster by country, host species or isolate, but most paralogues were 30-70 % similar, making meaningful alignments difficult. Some p44 paralogues from different isolates formed clusters of sequences that were more than 90 % similar to one another ('similarity groups'). The paralogues in each cluster were particularly similar in gene regions most likely to code for ligands. In the sample studied, 95 % of the similarity groups comprised paralogues from either USA or UK isolates only and occurred with greater frequency amongst paralogues from USA rather than UK isolates. These findings raise the hypothesis that sequences of paralogues in similarity groups may provide an index of adaptation of different 'strains' of A. phagocytophilum to specific reservoir hosts in different geographical locations, and any associations with infectivity for different species including humans.

Purification of ovine neutrophils and eosinophils: Anaplasma phagocytophilum affects neutrophil density.

Studies on the functions of ovine granulocytes require pure and functionally active populations of neutrophils and eosinophils. This report describes an improved technique for the separation of neutrophils and eosinophils from the peripheral blood of sheep infected with Anaplasma phagocytophilum and from normal sheep. After centrifugation and discarding the buffy coat layer, which contains the bulk of mononuclear cells, neutrophils with a high degree of purity (94.87 [+/-1.7]%, n=9) and good yield (69 [+/-9]%, n=9) were obtained by density gradient centrifugation on Percoll with a density of 1.09 g/ml (65%). However, this density was not suitable for neutrophils obtained from sheep during the peak period of A. phagocytophilum bacteraemia. Improved purity of infected neutrophils was obtained when the leucocytes were separated on Percoll with a density of 1.08 g/ml (55%). Relatively good purity of eosinophils was obtained when leucocytes from normal sheep were separated on Percoll with a density of 1.10 g/ml (70%). Ovine eosinophils formed a distinct band just below the band of mononuclear cells when a continuous Percoll gradient with a density of 1.10 g/ml was used. The purity of the eosinophils obtained was 87.7 (+/-12.5)% (n=6; range 64.1-97.6%), with a mean recovery rate of 61.9 (+/-20.3)%.

Transmission of Anaplasma phagocytophilum to Ixodes ricinus ticks from sheep in the acute and post-acute phases of infection.

A total of 60 sheep were exposed to Anaplasma phagocytophilum infection on an enclosed area of Ixodes ricinus-infested pasture in North Wales, United Kingdom, and rapidly acquired acute A. phagocytophilum infections detectable by PCR and blood smear examination. Of the ticks that had engorged in the previous instar on infected sheep, 52% of adult ticks and 28% of nymphs were PCR positive; a significant, 10-fold increase in prevalence compared to that of ticks that engorged on sheep preinfection was observed (P = 0.015). The likelihood that ticks were PCR positive, after feeding on the sheep and molting to the next instar, increased marginally with increasing numbers of infected neutrophils per milliliter of blood of their sheep host (P = 0.068) and increased significantly when they were collected from sheep carrying higher numbers of adult female ticks (P = 0.017), but increasing numbers of feeding nymphs had a significant negative effect on transmission (P = 0.049). The numbers of circulating neutrophils and of infected neutrophils also varied significantly with the numbers of ticks feeding on the sheep when the blood was collected. Our study suggests that ruminants are efficient reservoirs of A. phagocytophilum during the acute and post-acute phases of infection. The risk of ruminant-derived infections may, however, be strongly affected by variations in tick densities, which may influence transmission from acutely infected animals via effects on the numbers of infected cells in the blood and possibly by within-skin modulation of infection.

Cultivation of an ovine strain of Ehrlichia phagocytophila in tick cell cultures.

Ehrlichia phagocytophila (previously known as Cytoecetes phagocytophila) which causes tick-borne fever (TBF) in sheep and pasture fever in cattle in the UK and mainland Europe is transmitted by the temperate hard tick Ixodes ricinus. The disease in sheep is characterized by fever, leucopenia and immunosuppression. Studies on the pathogenesis and other aspects of the disease have been hampered because the organism has not been cultivated in continuous or primary cell culture systems. This paper describes the first successful cultivation of a European isolate of E. phagocytophila in two continuous cell lines, IDE8 and ISE6, derived from the temperate hard tick Ixodes scapularis. Once adapted to tick cell cultures the organism was serially sub-cultured in new cells by transferring small portions of infected cell suspension every 2 to 3 weeks. The identity of the organism was confirmed by polymerase chain reaction (PCR), with primers specific to the granulocytic ehrlichiae. Sequence analysis of the PCR products amplified from infected tick cells were shown to be identical with those amplified from the blood of sheep infected with the same strain of E. phagocytophila. A susceptible sheep inoculated with a third passage of the tick cell-adapted E. phagocytophila reacted with fever and rickettsiaemia 5 days later, thus satisfying Koch's postulates.

Prevalence of antibodies to Brucella spp. and risk factors related to high-risk occupational groups in Eritrea.

In a study of three high-risk occupational groups using Rose Bengal and complement fixation tests, the highest prevalence (7.1%) was found among dairy farm workers and owners in randomly selected dairy-cattle farms, followed by veterinary personnel (4.5%) and inhabitants in pastoralist areas (3.0%). There was no evidence for significant differences between the three populations. Among dairy farm workers, a higher risk was associated with the presence of sheep in the farm (OR = 13.2, CI = 2.2-76.7). In the pastoral area, a high risk was linked to having close contact with animals (OR = 6.32, CI = 0.88-infinity), while a reduced risk was seen for contact with cattle (OR = 0.18, CI = 0-1.30). Symptoms suggestive of brucellosis were more commonly observed among the dairy farm workers, mainly found in the highlands, than among the pastoralist area inhabitants, where malaria is prevalent. The study documents not only the presence of serological and clinical evidence of human brucellosis, but also risk factors related to it in Eritrea, for the first time.

IgG responses to salivary gland extract of Ixodes ricinus ticks vary inversely with resistance in naturally exposed sheep.

Enzyme-linked immunosorbent assay (ELISA) was used to investigate the antibody responses of control sheep, and sheep naturally exposed to Ixodes ricinus Linné (Acari: Ixodidae) ticks, to salivary gland extract (SGE) proteins of partially fed, adult I. ricinus. Comparisons between responses of control sheep and naturally infested sheep by Western blot analysis suggested that variations in IgG responses of I. ricinus-exposed sheep were mostly associated with specific responses to I. ricinus SGE antigens. Sheep IgG responses were positively related to the numbers of adult ticks feeding per sheep at the time samples were collected, were greater during the spring than the autumn periods of I. ricinus activity and were inversely related to sheep resistance to ticks measured by the weights of nymphal I. ricinus that engorged on the sheep. These findings suggest that sheep lose their resistance to ticks due to polarization of a Th1 type response to some tick antigens towards a Th2 type response when sheep are exposed to high, natural tick infestations, or to seasonal conditions of relative nutritional stress. Potential consequences for the epidemiology of tick-borne diseases are discussed.