Adaptive immunity to Anaplasma pathogens and immune dysregulation: implications for bacterial persistence

Anaplasma marginale is an obligate intraerythrocytic bacterium that infects ruminants, and notably causes severe economic losses in cattle worldwide. Anaplasma phagocytophilum infects neutrophils and causes disease in many mammals, including ruminants, dogs, cats, horses, and humans. Both bacteria cause persistent infection - infected cattle never clear A. marginale and A. phagocytophilum can also cause persistent infection in ruminants and other animals for several years. This review describes correlates of the protective immune response to these two pathogens as well as subversion and dysregulation of the immune response following infection that likely contribute to long-term persistence. I also compare the immune dysfunction observed with intraerythrocytic A. marginale to that observed in other models of chronic infection resulting in high antigen loads, including malaria, a disease caused by another intraerythrocytic 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.     

Tick-borne fever caused by Anaplasma phagocytophilum in Germany: first laboratory confirmed case in a dairy cattle herd

Four cows from North-West Germany have been diagnosed with tick-borne fever (TBF) based on the demonstration of morulae in neutrophilic granulocytes in their blood smears, positive signals in real-time PCR specific for Anaplasma phagocytophilum using DNA extracted from their buffy coats, and demonstration of specific antibodies in their sera using a commercially available immunofluorescence assay. Clinical findings included high fever, decreased milk production, lower limb edema with stiff walking, eye and nasal discharge, and depression. These signs developed about a week after the animals had been brought to the pasture for the first time in their life. All cows recovered after 5-15 days, although DNA of A.phagocytophilum could be detected by real-time PCR up to 6 weeks after onset of the disease. Considering the known prevalences of A.phagocytophilum in ticks in Germany and its detection in dogs and horses, we think that underdiagnosing of TBE in cattle is highly likely. Therefore TBF should be taken into account as differential diagnosis in case of high fever and/or a sudden decrease in milk production in pastured animals.     

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.     

Congenital infection with Anaplasma phagocytophilum in a calf in northern Germany

Anaplasma phagocytophilum is a Gram-negative, obligate intracellular tick-transmitted bacterium that replicates in neutrophils. It causes tick-borne fever (TBF) in sheep and cattle, but also elicits febrile disease in humans as well as in other domestic animals such as dogs, horses, and cats. Although increasingly recognized in Europe, the first laboratory-confirmed case of TBF in cattle from Germany has been published only recently. We here present the unusual case of an intrauterine transmission of A. phagocytophilum in a calf from northern Germany. To our knowledge, this is the first report of such an event occurring under field conditions in cattle.     

The effects of Ehrlichia (Cytoecetes) phagocytophila on the clinical chemistry of sheep and goats

Tick-borne fever (TBF) is a rickettsial disease of domestic and wild ruminants in temperate climates where the hard tick Ixodes ricinus is present. The disease is characterized by a high temperature and severe leukopenia. In the present study, the effects of TBF on the activity of serum alkaline phosphatase (ALP), and on the concentrations of plasma zinc, iron, total bilirubin, urea, creatinine and albumin were investigated by inoculating one group of eight sheep and one group of eight goats with the Old Sourhope (OS) strain of Ehrlichia (Cytoecetes) phagocytophila. All goats and sheep experimentally infected with E. phagocytophila reacted with fever, rickettsiaemia and leukopenia. The leukopenia was due to an acute lymphocytopenia and prolonged neutropenia. In both groups of animals. TBF was characterized by significant reductions in the activities of serum ALP and concentrations of plasma zinc, iron and albumin. However, there were significant increases in the concentrations of plasma total bilirubin, urea and creatinine in both species of animals. The reductions in ALP and iron were significantly more pronounced in sheep than in goats.     

Differential haematological effects of tick-borne fever in sheep and goats

Tick-borne fever (TBF) is a rickettsial disease of domestic and wild ruminants in temperate climates. It is characterized by high fever and severe leukopenia. In the present study, the possible difference in the severity of disease in sheep and goats was investigated by inoculating one group of eight goats and one group of eight sheep with the Old Sourhope (OS) strain of Ehrlichia (Cytoecetes) phagocytophila. All sheep and goats experimentally infected with E. phagocytophila reacted with fever and rickettsiaemia, but there were significant differences between goats and sheep in the severity of clinical disease, the duration and magnitude of fever, the magnitude of rickettsiaemia and the patterns of reduction in the number of total leucocytes. Sheep reacted with fever significantly earlier than goats and the febrile period lasted for a significantly longer period. In contrast, the magnitude of rickettsiaemia was significantly higher in goats than in sheep. Infection with TBF was characterized by a transient increase in the number of neutrophils, which was quickly followed by an acute reduction in the number of lymphocytes and a prolonged reduction in the number of neutrophils in both sheep and goats. In both groups of animals, infection with TBF was also characterized by significant reductions in the total number of red blood cells (RBCs), thrombocytes and packed cell volume (PCV) and the concentration of haemoglobin (Hb). However, the mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) were significantly increased in sheep only.     

Molecular detection of Anaplasma phagocytophilum in cattle and Ixodes persulcatus ticks

The tick-borne pathogen, Anaplasma phagocytophilum (A. phagocytophilum), the causative agent of human granulocytic anaplasmosis (HGA), is increasingly becoming a public health concern as an aetiological agent for emerging infectious disease. We found A. phagocytophilum infection in a pooled sample of field-collected Ixodes persulcatus (I. persulcatus) ticks from one district in Hokkaido, Japan. Thus, to further investigate the prevalence in field-collected ticks, we used PCR assays targeting the A. phagocytophilum gene encoding 44 kDa major outer membrane protein (p44) for screening of I. persulcatus ticks and samples from cattle from pastures. Out of the 281 I. persulcatus ticks, 20 (7.1%) were found to harbor A. phagocytophilum DNA. The infection rate for A. phagocytophilum in cattle was 3.4% (42/1251). In future studies, it will be necessary to investigate effects of the infection in order to understand its pathogenesis of A. phagocytophilum in domestic animals.     

Anaplasmosis in two dogs in France and molecular and phylogenetic characterization of Anaplasma phagocytophilum

Two dogs in France were diagnosed with Anaplasma phagocytophilum infection by real-time PCR. The most remarkable hematologic and biochemical findings were severe thrombocytopenia, mild neutrophilia, morulae in neutrophils, and increased serum concentration of the α2-globulin fraction detected by agarose gel electrophoresis of serum proteins. Using sequencing of the partial 16S rRNA and ankA genes, molecular characterization of the A. phagocytophilum strains showed that the organisms from both dogs were identical to the European strains isolated from horses and people. Based on phylogenetic analysis, the ankA gene was more discriminating than the 16S rRNA gene in distinguishing the majority of European and American strains of A. phagocytophilum infecting people and animals. Three isolates of A. phagocytophilum, 1 from Spain (cow) and 2 from Norway (sheep and deer), were external to the European and American clades.     

Leukocyte migration inhibition after experimental trypanosome infection in cattle

Blood serum from cattle experimentally infected with trypanosomes was tested for its activity influencing granulocyte migration. Pooled porcine granulocytes were used in the migration assay. The inhibitory migration activity observed in serum samples of trypanosome infected animals implies the presence of mediators of cellular immunity. Values of migration indices express reciprocal events of inhibitory and stimulating events in infected animals. This study allowed to follow at least some aspects of the very complex cellular immune system and its functioning. The observed differences in migration values in serum of each animal speak for an individual immunological capacity to defy trypanosome infections. The variability in occurrence of the first inhibitory activity after infection supports the view of individual responsiveness. Distinct differences in migration values were observed in Dahomey cattle after primary and secondary challenge with trypanosomes. Serum from reinfected animals exhibited a marked decrease in inhibitory activity as compared to samples obtained after first infection. These observations suggest a modulation of the individual immune response after multiple challenge with trypanosomes of the same strain. This study demonstrates the involvement of cell mediated immune responses to trypanosome infections.     

Molecular and serological evidence of Anaplasma phagocytophilum infection of farm animals in the Black Sea Region of Turkey

This study was designed to determine the presence and the prevalence of Anaplasma phagocytophilum infection in sheep and cattle in the Middle and Eastern Black Sea Regions of Turkey in which the potential vector, Ixodes ricinus, is widespread. Blood samples were collected from 720 sheep and 720 cattle from 6 provinces of the region, and used for detecting antibodies to A. phagocytophilum by indirect immunofluorescent antibody test (IFAT) and specific nucleic acids by a nested polymerase chain reaction (PCR). Blood smears were also prepared and examined microscopically for the presence of A. phagocytophilum-like organisms in polymorphonuclear cells. Of the animals examined, antibodies were detected in 110 (15.27%) cattle and 107 (14.86%) sheep and A. phagocytophilum-like organisms were detected in the blood of 73 (10.13%) cattle and 71 (9.86%) sheep. In addition, specific DNA was detected in the blood of 27 (14.75%) cattle and 22 (12.35%) sheep. The results obtained constitute the first molecular and serological evidence of A. phagocytophilum infection in sheep and cattle in the Black Sea Region of Turkey.     

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.     

Typical and atypical manifestations of Anaplasma phagocytophilum infection in dogs

Eighteen clinically ill dogs, naturally infected with Anaplasma phagocytophilum, were examined at a veterinary practice in Baxter, Minnesota. A clinical examination, complete blood cell count, enzyme- linked immunosorbent assay (ELISA) for A phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies and Dirofilaria immitis antigen, and a polymerase chain reaction test for A phagocytophilum DNA were obtained for all dogs. Physical examination findings included fever, arthropathy, lymphadenopathy, epistaxis, acute gastritis, cervical hyperpathia, and central nervous system dysfunction. Complete blood cell count abnormalities included thrombocytopenia, morulae in neutrophils, anemia, leukopenia, eosinopenia, lymphopenia, and monocytosis. Seroreactivity to A phagocytophilum was found in 61%, B burgdorferi antibodies in 17%, and D immitis antigen in 5% of the dogs. Fever, arthropathy, neurologic dysfunction, and epistaxis are clinical syndromes that can be associated with A phagocytophilum infection. Treatment with doxycycline resulted in rapid resolution of clinical signs in all dogs.     

Evidence of Anaplasma infections in European roe deer (Capreolus capreolus) from southern Spain

Anaplasma spp. (Rickettsiales: Anaplasmataceae) are tick-borne pathogens of veterinary and human importance. The wildlife hosts for these pathogens are not well characterized and may play an important role in the epidemiology of the disease. The objective of this research was to study the infection with A. marginale, A. ovis and A. phagocytophilum in free-ranging European roe deer (Capreolus capreolus) from Cádiz, Andalucía, Spain. Of 17 roe deer tested, 14 (82%) and 5 (29%) had antibodies reactive to Anaplasma spp. and A. phagocytophilum by competitive ELISA and indirect immunofluorescent antibody testing, respectively. Polymerase chain reaction and sequence analysis of Anaplasma major surface protein 4 (msp4) gene was conducted on blood samples from all roe deer examined. Nine (53%) animals had evidence of infection with A. ovis and 3 (18%) were positive for A. phagocytophilum. Concurrent infections were not detected. Despite the presence of A. marginale infections in cattle in the study site (36% msp4 PCR-positive animals), none of the msp4 amplicons from roe deer corresponded to A. marginale sequences. A. ovis msp4 sequences were identical to a genotype previously identified in sheep in Sicily, Italy. Two different A. phagocytophilum genotypes were identified in infected roe deer. This is the first report of roe deer naturally infected with A. ovis. These results demonstrate that roe deer are infected with A. ovis and A. phagocytophilum in Spain and suggest that this species may be involved in the natural cycle of these pathogens in this region, thus acting as potential reservoir for transmission to domestic and wild animals.     

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.     

Immunosuppressive effect of Trypanosoma congolense and Trypanosoma vivax on the secondary immune response of cattle to Mycoplasma mycoides subsp mycoides.

Trypanosoma congolense and T vivax infections in cattle were shown to have a suppressive effect on the secondary humoral immune response to M mycoides. The trypanosome infections caused an involution of the thymus. The secondary response was biphasic in nature and was unrelated to infection with trypanosomes.     

Ticks and tick-borne disease in Guatemalan cattle and horses

Blood samples and ticks were collected from 48 cattle and 74 horses from seven sites in the Peten region of Guatemala. Data on body condition, mucous membrane capillary refill time and tick infestation levels were recorded for each animal in the study. Horses had significantly higher levels of tick infestation than cattle, as well as poorer body condition scores. Seroprevalence of Babesia spp. was 95.8% for B. bovis in cattle, 89.6% for B. bigemina in cattle, and 92.7% for B. equi in horses. Seroprevalence of Anaplasma marginale in cattle was 87.5%, similar to reports in animals from other regions of Central America. This is the first time that A. phagocytophilum has been reported in animals from this region, with overall PCR-prevalence of 27.6% in cattle and horses, and seroprevalence of 28.4% (52% in cattle and 13% in horses). An agent was identified with serological cross-reactivity and close genetic relatedness to Ehrlichia ruminantium, but further testing confirmed that the agent in Guatemalan cows was not the agent of heartwater. Ticks were identified to species with the predominant species identified on cattle as Boophilus microplus and Amblyomma cajennense, while Anocentor nitens and A. cajennense were most commonly found on horses. Prevalence of infection, tick infestation levels, host factors and environmental data were analyzed for association; A. nitens was significantly associated with A. phagocytophilum prevalence by village.     

The influence of a low cobalt intake on the neutrophil function and severity of Ostertagia infection in cattle

Two trials involving housed cattle examined the effect of Co depletion and supplementation on immune status as assessed by the neutrophil function test which measures the ability of isolated neutrophils to kill the yeast Candida albicans. A third trial investigated the extent to which Co status influenced the severity of Ostertagia ostertagi infection. In the first two trials liveweight gains were unaffected until some 40-60 weeks on the low dietary Co intake despite very low serum vitamin B12 values being recorded after 10 weeks. However, the immune status as measured by the neutrophil function test was reduced within 10 weeks of commencing the low Co diet. On administration of Ostertagia ostertagi larvae, Co-depleted cattle showed a greater weight loss than Co-supplemented cattle but showed no difference in the length of the prepatent period, worm egg production or serum gastrin and pepsinogen concentrations. After anthelmintic treatment both groups showed a similar response. It is postulated that the lowered immune response of Co-depleted cattle resulted in the greater severity of the Ostertagia ostertagi infection.     

In vivo immunostimulatory effects of CpG oligodeoxynucleotide in cattle and sheep

Oligodeoxynucleotides (ODN) containing cytosine-phosphate-guanosine (CpG) motifs have been shown to activate the innate immune system and protect mice and chicken from bacterial and viral infections. Unfortunately, similar studies in other veterinary species are lacking. In this study we assessed the in vivo immunostimulatory effects of CpG ODN 2007, an ODN with previously demonstrated in vitro biological activity. The in vivo effects of ODN 2007 were compared in two closely related outbred species, sheep and cattle, to determine if there were common biological responses. We demonstrated that subcutaneous (s.c.) injection of the CpG ODN induces an acute phase response in the form of a transient fever, a mild transient increase in circulating neutrophils and elevated serum haptoglobin in both sheep and cattle. Sheep injected with CpG ODN also exhibited increased serum 2'5'-oligoadenylate (2'5'-A) synthetase activity, but no increase in serum 2'5'-A synthetase was detected in cattle. The ODN-induced responses were stronger in animals injected with CpG ODN formulated in 30% emulsigen than phosphate buffer saline (PBS) alone. These in vivo data demonstrate for the first time that a CpG ODN induces acute phase immunostimulatory responses in sheep and cattle. However, CpG ODN-induced antiviral effector molecule 2'5'-A synthetase was detected only in sheep but not in cattle.