Anaplasma phagocytophilum in Danish sheep: confirmation by DNA sequencing

Background

The presence of Anaplasma phagocytophilum, an Ixodes ricinus transmitted bacterium, was investigated in two flocks of Danish grazing lambs. Direct PCR detection was performed on DNA extracted from blood and serum with subsequent confirmation by DNA sequencing.

Methods

31 samples obtained from clinically normal lambs in 2000 from Fussingø, Jutland and 12 samples from ten lambs and two ewes from a clinical outbreak at Feddet, Zealand in 2006 were included in the study. Some of the animals from Feddet had shown clinical signs of polyarthritis and general unthriftiness prior to sampling. DNA extraction was optimized from blood and serum and detection achieved by a 16S rRNA targeted PCR with verification of the product by DNA sequencing.

Results

Five DNA extracts were found positive by PCR, including two samples from 2000 and three from 2006. For both series of samples the product was verified as A. phagocytophilum by DNA sequencing.

Conclusions

A. phagocytophilum was detected by molecular methods for the first time in Danish grazing lambs during the two seasons investigated (2000 and 2006).     

Chemoprophylaxis of Anaplasma ovis infection in sheep with a long-acting oxytetracycline

The chemoprophylactic efficacy of long-acting oxytetracycline was determined in 13 susceptible ewes infected with Anaplasma ovis. The drug was administered intramuscularly at a dose rate of 20 mg kg-1 body weight during the prepatent period. When the ewes were exposed to an equivalent homologous challenge on Day 45 post-infection, each showed a mild or inapparent reaction. The host reactions, i.e., body temperature, parasitaemia, packed-cell volume (PCV), haemoglobin, humoral and cell mediated immunity (CMI) were also studied. The results indicate that the use of oxytetracycline during the incubation period would minimise clinical signs of an Anaplasma infection; this may be due to an increasing CMI response.     

Superinfection occurs in Anaplasma phagocytophilum infected sheep irrespective of infection phase and protection status

Background

Anaplasma phagocytophilum infection in domestic ruminants is widespread in the coastal areas of southern Norway. The bacteria may persist in mammalian hosts. Several genetic variants of A. phagocytophilum exist. In the present study, we investigate whether superinfection occurs in the acute and persistent phase of the infection.

Methods

Five-month-old lambs of the Norwegian Dala breed were experimentally infected with two 16S rRNA gene variants of A. phagocytophilum, i.e. A. phagocytophilum variant 1 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"M73220","term_id":"148293","term_text":"M73220"}}M73220) and variant 2 (GenBank acc. no. {"type":"entrez-nucleotide","attrs":{"text":"AF336220","term_id":"13325085","term_text":"AF336220"}}AF336220). Eighteen lambs were used, two lambs in each group. Eight groups were experimentally inoculated with either variant 1 or 2 on day 0. Six of these groups were then challenged with the other variant on either days 7, 42 or 84, respectively. One group was left uninfected. The occurrence of A. phagocytophilum in blood samples was determined using semi-nested PCR analysis and gene sequencing. Specific antibodies were measured by an indirect immunofluorescence antibody assay (IFA).

Results

A. phagocytophilum variant 1 and 2 differed significantly with regards to clinical reaction and cross-immunity in infected lambs. Both variants were found in the blood after challenge. However, variant 1 was detected most frequently.

Conclusion

The present experiment indicates that superinfection of different genotypes occurs during the acute as well as the persistent phase of an A. phagocytophilum infection, even in lambs protected against the challenged infection.     

Differential expression of inflammatory and immune response genes in sheep infected with Anaplasma phagocytophilum

Anaplasma phagocytophilum infects a wide variety of host species and causes the diseases tick-borne fever (TBF) in ruminants and granulocytic anaplasmosis in humans, horses and dogs. TBF in sheep has become one of the more prevalent tick-borne diseases in some regions of Europe. A. phagocytophilum infection modifies host gene expression and immune response. The objective of this research was to characterize differential gene expression in sheep experimentally and naturally infected with A. phagocytophilum by microarray hybridization and real-time RT-PCR. The results of these studies demonstrated in sheep the activation of inflammatory and innate immune pathways and the impairment of adaptive immunity during A. phagocytophilum infection. The characterization of the genes and their expression profiles in sheep in response to A. phagocytophilum infection advances our understanding of the molecular mechanisms of pathogen infection and the pathogenesis of TBF. Collectively, these results expand current information on the mammalian host response to A. phagocytophilum infection.     

Validation of an Anaplasma marginale cELISA for use in the diagnosis of A. ovis infections in domestic sheep and Anaplasma spp. in wild ungulates

A commercially available (cELISA) kit for diagnosing Anaplasma marginale infection in cattle was validated for diagnosing A ovis infection in sheep using the bovine serum controls as supplied by the manufacturer (BcELISA) and sheep serum controls from pathogen-free sheep (OcELISA). True positives were identified using two previously established assays, a nested PCR (nPCR) test and an indirect immunofluorescent assay (IFA). The BcELISA was also applied to sera from various species of wild ruminants, comparing the results with the IFA. Receiver operating characteristic (ROC) analysis indicated that the predicted threshold inhibition for the BcELISA was 19.2. The sensitivity for the BcELISA was 98.2% and the specificity was 96.3%. The predicted threshold inhibition decreased to 14.3 for the OcELISA; the sensitivity was 96.5% and the specificity was 98.1%. There was >/=90% concordance between IFA and nPCR, as well as between the BcELISA at 19% inhibition cutoff and either IFA or PCR. Concordance between the cELISA and IFA using sera from elk, mule deer, bighorn sheep, pronghorn antelope, and black-tailed deer ranged from 64% to 100%. This commercially available cELISA test kit can be used very effectively to test domestic sheep for infection with A. ovis using the kit-supplied controls (i.e. the BcELISA) and a 19% inhibition cutoff; the kit may also be useful for detecting intra-erythrocytic Anaplasma infections in wild ruminants.     

Modulation of leukocyte populations and immune responses in sheep experimentally infected with Anaplasma (formerly Ehrlichia) phagocytophilum

Anaplasma phagocytophilum infection in sheep is characterized by an immune suppression as indicated by impaired antibody response, reduced lymphocyte response and reduced oxidative burst. The effect of A. phagocytophilum infection on leucocyte populations, especially lymphocytes, was therefore investigated in six sheep experimentally infected with A. phagocytophilum, and compared with leucocyte populations from control animals.To investigate the ability of the infection to interfere with the cellular and humoral responses to specific antigens, the animals were vaccinated with commercial vaccines at the time of experimental infection, and monitored for 56 days.There were reduced percentages of gammadelta T-cells and CD4+ T-cells in peripheral blood of infected animals throughout the study period, and these cell populations showed a down-regulation of CD25 expression; while there was a relative increase in CD8+ T-cells. The reduction in CD25+ gammadelta T-cells involved a subpopulation of WC1+ gammadelta T-cells. During the first 2 weeks of the study there were reduced percentages of B-cells and leukocytes expressing MHC II and CD11b, though this decrease changed to a relative increase later in the study. The relative reductions in leucocyte populations corresponded with the observed leucopenia during the first 3 weeks post-infection, which involved lymphocyte, neutrophil and eosinophil subsets [Vet. Immunol. Immunopathol. 86 (2002) 183]. There was a reduced expression of CD11b and CD14 on granulocytes during the first 2 weeks of the study, which corresponded with the previously reported leucopenia involving neutrophils and eosinophils. Antibody responses to vaccines, lymphocyte in vitro proliferative responses to antigens and mitogens, and in vitro IFN-gamma responses to antigens were reduced up to 4 weeks after infection.     

An Anaplasma centrale DNA probe that differentiates between Anaplasma ovis and Anaplasma marginale DNA

An Anaplasma centrale genomic library was constructed in pUC13. Two clones pAC5 and pAC137 hybridising to A. centrale and A. marginale DNA were isolated from this library. One of these, pAC5, also hybridised to DNA from A. ovis. The total insert of pAC5 was subcloned into pBR322. This subclone, pAC5-12, could detect 1 ng A. centrale, 0.5 ng A. marginale and 3.9 ng A. ovis DNA. The hybridisation pattern obtained with pAC5-12 on digests of A. centrale, A. marginale and A. ovis DNA suggests that this probe detects EcoR1 and Hind111-polymorphisms. Probe pAC5-12 could detect A. ovis DNA in 36% of blood samples tested compared to the 33% detectability obtained with microscopy.     

Evaluation of sequential coinfection with Anaplasma phagocytophilum and Anaplasma marginale in cattle

OBJECTIVE: To determine whether sequelae of infection differed among single versus double infection with Anaplasma phagocytophilum or Anaplasma marginale, with and without tick salivary extract, in cattle. ANIMALS: Eighteen 13-month old steers. PROCEDURES: Treatment groups of 3 cattle each included A marginale inoculated ID followed on day 35 by A phagocytophilum without tick saliva, A phagocytophilum followed on day 10 by A marginale without tick saliva, A marginale followed on day 35 by A phagocytophilum with tick saliva, A phagocytophilum followed on day 10 by A marginale with tick saliva, tissue culture control injection, and tick saliva control injection. Infection was monitored via clinical observations, CBC, serologic testing, and PCR analysis of blood and tissues. RESULTS: Infected cattle had significantly reduced weight gain. Anemia occurred 25 to 32 days after A marginale infection, which was attenuated by tick saliva. Parasitism was greater if cattle had not previously been inoculated with A phagocytophilum. Nine of the 12 treated cattle had positive results of PCR analysis for A phagocytophilum from at least 1 blood sample. Five tissue samples had positive results of PCR analysis for A phagocytophilum; PCR results for A marginale were positive in spleen, lung, lymph node, heart, and ear skin of infected cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated an important biological interaction between A marginale and A phagocytophilum infection as well as with tick saliva in disease kinetics and severity in cattle, which may be important for interpretation of diagnostic tests and management of disease in areas where both pathogens occur.     

Characterization of Anaplasma phagocytophilum and A. ovis infection in a naturally infected sheep flock with poor health condition

Anaplasma species are transmitted by ticks and cause diseases in humans and animals. These pathogens infect sheep, an economically important domestic animal worldwide. The current study was designed to characterize in 200 animals the infection with Anaplasma phagocytophilum and Anaplasma ovis and the genetic diversity of A. ovis strains collected from a naturally infected sheep flock with poor health condition. Sheep had 98% seroprevalence to Anaplasma spp. antibodies. PCR results confirmed the presence of A. phagocytophilum and A. ovis DNA in 11.5% and 37% of the sheep, respectively. Concurrent infections were detected in 6.5% of the sheep. Seventy-one adult ticks were collected from 45 sheep with infestations ranging from one to 15 ticks per animal. The analysis of A. ovis msp4 sequences demonstrated a previously unreported polymorphism for this pathogen with 17 different haplotypes in infected sheep. These results demonstrated that, although A. ovis msp4 haplotypes may be less variable when compared with Anaplasma marginale and A. phagocytophilum strains on a global scale, genetic polymorphisms occur in this locus in strains obtained from an infected sheep flock with poor health condition.     

利用套式PCR检测牛羊乏质体

为同时检测和鉴定牛边缘乏质体、中央乏质体及绵羊乏质体,根据这3种病原体的msp4基因核苷酸序列,自行设计、合成了针对3种乏质体的2对通用引物,及分别针对三者的特异引物,通过PCR条件优化,建立了检测乏质体及分别鉴定3种乏质体的套式PCR方法,并与OIE推荐的msp5半套式PCR比较.结果显示:该方法对牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫、伊氏锥虫均未扩增出特异性片段.套式PCR检测乏质体DNA量为0.2 pg(相当于6个感染红细胞).检测l 119份来自6个不同地区的奶牛、肉牛、水牛及羊的临床样品,阳性106份,经鉴定边缘乏质体46份,中央乏质体15份,绵羊乏质体35份,混合感染中央乏质体和绵羊乏质体4份,混合感染边缘乏质体和绵羊乏质体3份,混合感染边缘乏质体和中央乏质体3份.首次在分子生物学水平证明中央乏质体存在于中国.同时,证明牛可以混合感染边缘乏质体和中央乏质体或绵羊乏质体,以及混合感染中央乏质体和绵羊乏质体.上述848份样品用OIE推荐的msp5半套式PCR同时检测,两者符合率为98.5％(835/848).检测结果表明,msp4套式PCR特异、敏感,可用于边缘乏质体、中央乏质体、绵羊乏质体的检测和鉴定.     

DNA probes for the detection of Anaplasma centrale and Anaplasma marginale

Anaplasmosis can be diagnosed either by immunological techniques or by direct microscopic examination of blood smears. Both methods are time-consuming and labour intensive. The use of DNA probes in an hybridization assay may simplify the diagnosis of anaplasmosis in cattle and sheep. A genomic DNA library of Anaplasma centrale was constructed in an expression vector and screened to detect clones containing A. centrale DNA. Four probes which hybridized to A. centrale and Anaplasma marginale DNA were isolated. One of these (AC-1) hybridized only to A. centrale DNA, whereas AC-2, AC-3 and AC-4 could detect DNA from both A. centrale and A. marginale. Probes AC-1 and AC-2 could detect 127 ng and 8 ng DNA respectively, while AC-3 and AC-4 detected 64 ng A. centrale DNA.     

Identification of immunodominant polypeptides common between Anaplasma centrale and Anaplasma marginale

High titered antibody from rabbits immunized with Anaplasma centrale or from cattle recovered from A. centrale infection bound predominantly to several 33-36 kDa polypeptides present in both A. centrale and the Israel-NT isolate of Anaplasma marginale. High titered bovine antibody against the Israel-NT isolate of A. marginale also reacted predominantly with A. centrale polypeptides in this size range. The immunodominance of the 33-36 kDa polypeptides and their cross-reactivity indicate that these shared epitopes may be primarily responsible for the cross-protective immunity between A. centrale and A. marginale.     

Test of the sheep ked Melophagus ovinus (L) as a vector of Anaplasma ovis Lestoquard

The sheep ked Melophagus ovinus was evaluated as a possible vector of Anaplasma ovis. In 4 tests, 45 to 293 keds were transferred from sheep with acute anaplasmosis, low level parasitemia, or carrier state of anaplasmosis to individual splenectomized ewes. Keds were confined in stockinette cages attached to the dorsal midline of the recipient hosts for 10 days. Throughout the 90-day observation periods after the keds were transferred, no clinical, serologic, or hematologic evidence of anaplasmosis was detected in any of the ked-recipient ewes. Results indicate that sheep keds probably are not mechanical or biological vectors of the ovine anaplasmosis organism.     

Frozen and fresh Anaplasma centrale vaccines in the protection of cattle against Anaplasma marginale infection

The immunity induced by frozen and fresh Anaplasma centrale vaccines against anaplasmosis caused by A. marginale was tested in 12-month old Friesian steers. A. centrale parasitaemia occurred in all cattle inoculated with both types of vaccine. The average maximal decrease in PCV for the frozen and fresh vaccines was 41.0 and 40.3% respectively. All cattle recovered spontaneously. Vaccinated and control steers of the same age were challenged six months later with doses of 10(6), 10(7) or 10(8) A. marginale organisms. Vaccinated cattle showed average maximal A. marginale parasitemia of 1.2-4.0 versus 10.3-12.0% in control cattle. The average maximal decrease in packed cell volume (PCV) was 33.1 and 30.0% for steers vaccinated with frozen or fresh vaccine, respectively, and 57.4% for the non-vaccinated steers. All vaccinated cattle recovered spontaneously from the A. marginale infection while 7 out of 8 control steers required specific treatment. It thus appears that both frozen and fresh A. centrale vaccines are equally capable of inducing partial protection against infection with A. marginale and of preventing severe red blood cell destruction.     

Analysis of protein compositions and surface protein epitopes of Anaplasma centrale and Anaplasma marginale.

Protein composition was compared and epitopes were analyzed among the isolates of Anaplasma centrale and A. marginale by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting using bovine antisera and monoclonal antibodies, and enzyme-linked immunosorbent assay. Common and unique proteins were found among the isolates. All isolates tested had a major surface protein with an apparent molecular weight of 38 to 40 kilodalton which had slight molecular size variations between species. This protein was also a dominant immunogen to the host. At least two species-common epitopes, one of which might contain carbohydrate(s), were present on the major surface protein. One species-specific epitope was identified on the major surface protein of A. marginale isolates.     

A comparative study of clinical manifestations,haematological and serological responses after experimental infection with Anaplasma phagocytophilum in two Norwegian sheep breeds

Background

It has been questioned if the old native Norwegian sheep breed, Old Norse Sheep (also called Norwegian Feral Sheep), normally distributed on coastal areas where ticks are abundant, is more protected against tick-borne infections than other Norwegian breeds due to a continuously high selection pressure on pasture. The aim of the present study was to test this hypothesis in an experimental infection study.

Methods

Five-months-old lambs of two Norwegian sheep breeds, Norwegian White (NW) sheep and Old Norse (ON) sheep, were experimentally infected with a 16S rRNA genetic variant of Anaplasma phagocytophilum (similar to GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"M73220","term_id":"148293"}}M73220). The experiment was repeated for two subsequent years, 2008 and 2009, with the use of 16 lambs of each breed annually. Ten lambs of each breed were inoculated intravenously each year with 0.4 ml A. phagocytophilum-infected blood containing approximately 0.5 × 10⁶ infected neutrophils/ml. Six lambs of each breed were used as uninfected controls. Half of the primary inoculated lambs in each breed were re-challenged with the same infectious dose at nine (2008) and twelve (2009) weeks after the first challenge. The clinical, haematological and serological responses to A. phagocytophilum infection were compared in the two sheep breeds.

Results

The present study indicates a difference in fever response and infection rate between breeds of Norwegian sheep after experimental infection with A. phagocytophilum.

Conclusion

Although clinical response seems to be less in ON-lambs compared to NW-lambs, further studies including more animals are needed to evaluate if the ON-breed is more protected against tick-borne infections than other Norwegian breeds.     

Transformation of Anaplasma marginale

The tick-borne pathogen, Anaplasma marginale, has a complex life cycle involving ruminants and ixodid ticks. It causes bovine anaplasmosis, a disease with significant economic impact on cattle farming worldwide. The obligate intracellular growth requirement of the bacteria poses a challenging obstacle to their genetic manipulation, a problem shared with other prokaryotes in the genera Anaplasma, Ehrlichia, and Rickettsia. Following our successful transformation of the human anaplasmosis agent, A. phagocytophilum, we produced plasmid constructs (a transposon bearing plasmid, pHimarAm-trTurboGFP-SS, and a transposase expression plasmid, pET28Am-trA7) designed to mediate random insertion of the TurboGFP and spectinomycin/streptomycin resistance genes by the Himar1 allele A7 into the A. marginale chromosome. In these trans constructs, expression of the fluorescent and the selectable markers on the transposon, and expression of the transposase are under control of the A. marginale tr promoter. Constructs were co-electroporated into A. marginale St. Maries purified from tick cell culture, and bacteria incubated for 2 months under selection with a combination of spectinomycin and streptomycin. At that time, ≤1% of tick cells contained colonies of brightly fluorescent Anaplasma, which eventually increased to infect about 80–90% of the cells. Cloning of the insertion site in E. coli and DNA sequence analyses demonstrated insertion of the entire plasmid pHimarAm-trTurboGFP-SS encoding the transposon in frame into the native tr region of A. marginale in an apparent single homologous crossover event not mediated by the transposase. Transformants are fastidious and require longer subculture intervals than wild type A. marginale. This result suggests that A. marginale, as well as possibly other species of Anaplasma and Ehrlichia, can be transformed using a strategy of homologous recombination.