Cloning and expression of the gene encoding the major surface protein 5 (MSP5) of Anaplasma phagocytophilum and potential application for serodiagnosis

BACKGROUND: Anaplasma phagocytophilum (formerly known as the human granulocytic ehrlichia, Ehrlichia equi and Ehrlichia phagocytophila) is an obligate intracellular organism causing clinical disease in humans and various species of domestic animals. OBJECTIVES: The objectives of this investigation were to sequence and clone the major surface protein 5 (MSP5) of A phagocytophilum and to evaluate the suitability of this antigen in the serologic diagnosis of anaplasmosis in humans and dogs. METHODS: The msp5 gene of A phagocytophilum was sequenced, cloned, and expressed in Escherichia coli. The predicted amino acid sequence homology of the various MSP5/major antigenic protein 2 orthologs was compared among various Anaplasma and Ehrlichia species. Recombinant MSP5 of A phagocytophilum was used in an ELISA to detect antibodies in serum samples from humans and dogs infected with the organism. RESULTS: Serum samples from 104 individuals previously diagnosed with A phagocytophilum infection, as well as samples from clinically healthy humans, were tested. In addition, multiple samples from 4 dogs experimentally infected with 2 different geographic isolates of A phagocytophilum and 5 dogs naturally infected with a Swiss isolate were tested using ELISA. Using this group of immunofluorescent antibody test-positive and immunofluorescent antibody test-negative samples, we found the overall agreement between assays to be >90%. CONCLUSIONS: These results indicate that recombinant MSP5 has potential for use as a diagnostic test antigen to detect infection with A phagocytophilum in both dogs and humans. However, sequence similarities among orthologs of MSP5 in related species of anaplasma and ehrlichia suggest that cross-reactivity among these pathogens is likely if the entire peptide is used as a test antigen.     

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.     

Molecular evidence of tick-transmitted infections in dogs and cats in the United Kingdom

PCR analysis was used to determine the prevalence of tick-transmitted infections in 120 systemically ill dogs and 60 cats recruited over a period of three months from 52 veterinary practices in the UK. The animals had not travelled outside the UK and had one or more of the following clinical criteria: acute or recurrent pyrexia, anaemia and/or thrombocytopenia, polyarthritis/muscle pain, splenomegaly/lymphadenopathy, and intraocular inflammation with systemic signs. Blood samples from the animals were tested for the presence of DNA from Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum by using simple PCR targeting. B. burgdorferi sensu lato was detected in five dogs and two cats, and A. phagocytophilum was detected in one dog and one cat. These results provide the first molecular evidence of naturally occurring B. burgdorferi sensu lato infection in cats in the UK and confirm that A. phagocytophilum infection is present in cats. There were no statistically significant associations between the infections and the clinical signs shown by the dogs and cats.     

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.     

Anaplasma phagocytophilum in dogs in Germany

A total number of 111 dogs were included in the present prospective study investigating the prevalence of Anaplasma phagocytophilum in dogs in Germany. Dogs were divided into two groups. Dogs of group 1 (n = 49) showed clinical and/or haematological signs seen in infections with A. phagocytophilum, whereas those of group 2 (n = 62) did not have any evidence of anaplasmosis. For each dog, an A. phagocytophilum 16S rRNA-nested polymerase chain reaction (PCR) of ethylenediaminetetraacetic acid (EDTA)-anticoagulated whole blood analysis, a microscopic evaluation of a buffy coat and a serum indirect fluorescent antibody test (IFAT) were performed. Forty-eight seroreactive dogs were identified altogether, which amounts to an overall point prevalence of 43.2%. There was no significant difference between the seroreactivity to A. phagocytophilum antigens among group 1 (44.9%) and 2 (41.9%) (P > 0.5). Seven dogs (6.3%) had positive PCR results. All of them were seroreactive. Six belonged to group 1. Morulae in neutrophilic granulocytes were found in two dogs of group 1 but in none of group 2. Both dogs were seroreactive. Very high antibody titres (> or =1:1024) were detected significantly more frequently in dogs with clinical signs attributable to infection with A. phagocytophilum (group 1) than in those without (group 2) (P < 0.001). There was no significant correlation of overall positives or antibody titres to age, breed, sex, or whether the dogs were family or working dogs. Dogs with high tick infestation were significantly more often seroreactive to A. phagocytophilum than those with no or low tick infestation (P = 0.007). In conclusion, there seems to be a high risk of infection with A. phagocytophilum in Germany. Results of this study suggest that severe illness solely caused by A. phagocytophilum may be possible although definitive evidence does not exist. Very high antibody titres (>1:1024) may be associated with clinical anaplasmosis.     

Clinical presentation of 26 anaplasma phagocytophilum-seropositive dogs residing in an endemic area

Anaplasma (A.) phagocytophilum, the etiological agent of canine granulocytic anaplasmosis, is capable of inciting moderate to severe clinical disease in a variety of mammals and is endemic in the upper midwest. The purpose of this study was fourfold: to describe the range of clinical signs in dogs seropositive to A. phagocytophilum; to examine the prevalence of immune-mediated hemolytic anemia (IMHA) in this population; to evaluate whether specific clinical signs were associated with coexposure to Borrelia (B.) burgdorferi in actively infected dogs; and to determine whether clinical response to doxycycline was complete in treated dogs. Medical records of dogs seropositive for A. phagocytophilum were reviewed retrospectively. Peripheral blood smears were also reviewed retrospectively for granulocytic Anaplasma morulae. Lethargy (81%), inappetence (58%), and lameness (50%) were the most common clinical signs, followed by fever (46%). Thrombocytopenia was the most common laboratory abnormality, and IMHA was diagnosed in three dogs. Dogs that were thrombocytopenic and had antibodies to both A. phagocytophilum and B. burgdorferi had a median platelet count of 51,000/μL (range 20,000 to 171,000/μL), which was significantly lower than the count in dogs with antibodies only to A. phagocytophilum (P=0.04). Some dogs had an apparent relapse of clinical signs after an appropriate course of doxycycline. Testing for A. phagocytophilum by polymerase chain reaction, serum antibody assays, and/or blood smear evaluation should be considered in dogs with IMHA, cough, or epistaxis and that reside in A. phagocytophilum-endemic areas. If moderate to severe thrombocytopenia is present, testing for concurrent B. burgdorferi infection may be warranted.     

The ability of a topical novel combination of fipronil, amitraz and (S)-methoprene to protect dogs from Borrelia burgdorferi and Anaplasma phagocytophilum infections transmitted by Ixodes scapularis

Healthy, purpose-bred laboratory beagle dogs that had not been exposed to ticks and were seronegative for Borrelia burgdorferi and Anaplasma phagocytophilum were randomly assigned to four groups of eight dogs each. Control group 1 was not treated. Groups 2, 3 and 4 were treated with a single topical application of a new formulation of fipronil, amitraz and (S)-methoprene (CERTIFECT?, Merial Limited, GA, USA) at 28, 21 or 14 days prior to tick infestation, respectively. Each dog was infested with 25 female and 25 male field-collected adult Ixodes scapularis ticks that had infection rates of 66% for B. burgdorferi sensu stricto and 23% for A. phagocytophilum, as determined by polymerase chain reaction. Two and five days after tick infestation, control dogs had an average of 9.5 and 13.9 attached adult female ticks, respectively, whilst the 24 treated dogs remained tick-free aside from a single tick on the 2nd day after infestation. Serial serological tests demonstrated that the ticks successfully infected 8/8 control dogs with B. burgdorferi and co-infected 6/8 with A. phagocytophilum. B. burgdorferi infection also was confirmed in most control dogs by culture (6/8) and PCR (7/8) of skin biopsies. In contrast, CERTIFECT protected all 24 treated dogs against infection by both B. burgdorferi and A. phagocytophilum, as demonstrated by their negative serological tests throughout the study and the absence of any positive skin biopsy culture or PCR in these dogs.     

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.     

Infection of endothelial cells with Anaplasma marginale and A. phagocytophilum

Anaplasma marginale and A. phagocytophilum are obligate intracellular, tick-borne pathogens that target erythrocytes and neutrophil granulocytes, respectively. Because ticks do not directly tap blood vessels, an intermediate tissue may mediate infection of blood cells. We considered that vascular endothelium interacts with circulating blood cells in vivo, and could be involved in pathogenesis and dissemination of the organisms. We used light and electron microscopy and immune labeling to show that A. phagocytophilum invaded rhesus (RF/6A), human (HMEC-1, MVEC), as well as bovine (BCE C/D-1b) endothelial cell lines, whereas A. marginale infected rhesus and bovine endothelial cells. A. marginale formed large intracellular inclusions that appeared smooth and solid at first, and subsequently coalesced into discrete granules. A. phagocytophilum formed numerous smaller inclusions in each cell. Within 1-3 weeks, the monolayers were destroyed, and lysed cultures were diluted onto fresh monolayers. Electron microscopy demonstrated uneven distribution of A. marginale inside large inclusions, with reticulated forms grouped more tightly than denser cells, whereas in A. phagocytophilum individual organisms appeared more evenly spaced. Specific polyclonal and monoclonal antibodies both labeled A. marginale and A. phagocytophilum in endothelial cells, and oligonucleotide primers complimentary to either A. marginale or A. phagocytophilum amplified their expected target from these cultures. In conclusion, we demonstrate that relevant microvascular endothelium is susceptible to anaplasmas in vitro and may present a link that could explain development of the immune response and persistent infection.     

Seroprevalence of Anaplasma phagocytophilum infection in dogs in Germany

To determine the prevalence of Anaplasma phagocytophilum in dogs in Germany serum samples from 1124 dogs that were under suspicion of having anaplasmosis were examined. The samples were tested by an indirect immunofluorescence test (OFT) for antibodies to A. phagocytophilum. The geographical origin of positive cases were analysed with an geographic information system. Antibodies to A. phagocytophilum were found in 563 (50.1%) of the tested dogs. 166 dogs came from Saarland, 161 from North Rhine-Westphalia, 134 from Baden-Wuerttemberg, 33 from Bavaria, 22 from Rhineland-Palatinate, 11 from Hamburg, 10 from Brandenburg, 9 from Lower Saxony, 8 from Hesse and Berlin respectively and 1 from Schleswig-Holstein. Clinical signs and laboratory findings of 26 seropositive dogs were analysed. Those dogs showed a low haematocrit, thrombocytopenia and leucocytoses as well as higher values for alkaline phosphatase, ALAT und bilirubin. The clinical signs were lameness in 13 dogs, lethargy in 5, and uveitis in 3 dogs. Rhinitis and lymphadenopathy was found in 2 dogs and retinal detachment with blindness in 1 dog.     

Experimental infection of C3H/HeJ mice with the NY18 isolate of Anaplasma phagocytophilum

Human granulocytic anaplasmosis (HGA), an emerging disease of public health concern in many areas of the world, is caused by Anaplasma phagocytophilum. Small animal models of A phagocytophilum in laboratory mice have been developed and used to study the pathogenesis of HGA. In this study, we characterized the pathologic changes in acute infection of C3H/HeJ mice experimentally infected with the NY18 isolate of A phagocytophilum. Although no clinical signs were noted, acute infection was associated with gross splenomegaly, microscopic inflammatory lesions in the lung and liver, hyperplastic lesions on the spleen, and clinical pathology abnormalities including neutropenia and monocytosis. This study emphasizes the use of well-defined animal models as a valuable tool for the study of A phagocytophilum infections.     

Molecular Evidence for Persistence of Anaplasma phagocytophilum in the Absence of Clinical Abnormalities in Horses after Recovery from Acute Experimental Infection

Background: Anaplasma phagocytophilum infects several mammalian species, and can persist in sheep, dogs, and calves. However, whether this organism persists in horses or induces long-term clinical abnormalities is not known.
Objectives: To evaluate whether A. phagocytophilum can persist in horses and to document clinical findings for 3 months after complete recovery from acute disease.
Animals: Five clinically normal adult horses that had recovered spontaneously from experimentally induced acute disease caused by a Swedish equine isolate of A. phagocytophilum .
Methods: Horses were monitored for up to 129 days post inoculation (PI) by daily clinical examination and at least alternate day blood sampling for evidence of A. phagocytophilum on polymerase chain reaction (PCR) and blood smears. All horses were euthanized and underwent postmortem examination.
Results: All horses were periodically PCR positive after recovery from acute infection. Before day 66 PI 2 horses were persistently PCR negative whereas 3 horses were intermittently PCR positive. Subsequently, 4 of 5 horses were intermittently PCR positive, particularly after stress mimicking interventions. One animal was positive immediately before postmortem examination. Clinical abnormalities related to persistence of anaplasma were not observed. No specific changes were found at postmortem examination, and all sampled tissues from all horses were negative on PCR for A. phagocytophilum .
Conclusions and Clinical Importance: Infection with A. phagocytophilum can persist in the horse for at least 129 days. However, the continued presence of the organism is not associated with detectable clinical or pathological abnormalities.     

Clinical features of canine granulocytic anaplasmosis in 18 naturally infected dogs

BACKGROUND: Anaplasma phagocytophilum, the causative agent of canine granulocytic anaplasmosis (CGA), is a Gram-negative intracellular organism transmitted by ixodid ticks. Thus far, only a few clinical studies evaluating dogs with CGA have been published. OBJECTIVES: Evaluation of dogs naturally infected with A. phagocytophilum in which known co-infections were excluded. ANIMALS: Eighteen dogs with CGA. METHODS: Prospective study. The diagnosis of CGA was based on a positive PCR test result; dogs with co-infections were excluded. History, clinical findings, CBC, clinical biochemistry, infectious disease screening, diagnostic imaging, and the course of disease were evaluated. RESULTS: CGA was diagnosed based on a positive PCR test for A. phagocytophilum; 10 dogs also had morulae in neutrophils. Six of 18 dogs were seronegative to A. phagocytophilum, the others were seropositive. All dogs were acutely ill. The most common clinical findings were lethargy, inappetence, fever, and splenomegaly. Abnormal laboratory results included thrombocytopenia, anemia, lymphopenia, hypoalbuminemia, and abnormally high plasma alkaline phosphatase activity. In 6 of 10 dogs tested, the platelet-bound antibody test was positive; Coombs' test was negative in 9 dogs. All dogs were treated with doxycycline and recovered. PCR testing as well as blood smear analysis for morulae were negative in 14 tested dogs 2-8 weeks after beginning treatment. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical findings in dogs with CGA were nonspecific. Positive platelet-bound antibody test results suggest immune-mediated platelet destruction as an important pathogenic mechanism. With correct diagnosis and treatment, prognosis is good.     

Prevalence of antibodies against Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum and their clinical relevance in dogs in Munich, Germany

Although prevalences of antibodies against Borrelia (B.) burgdorferi sensu lato (sl) and Anaplasma (A.) phagocytophilum have been reported to be high in the German dog population, the importance of the diseases caused by both agents is still not well characterized in a field situation.The aim of this study was (1) to determine the prevalence of antibodies to B. burgdorferi sl and A. phagocytophilum in dogs in Munich, Germany, and (2) to assess the clinical presentation and laboratory values of antibody-positive dogs and compare them to a negative control group. In total, 448 randomly selected dogs were screened for antibodies to B. burgdorferi sl and A. phagocytophilum with the SNAP 4Dx assay (IDEXX, Laboratories, Inc., USA). Dogs carrying antibodies against B. burgdorferi sl and/or A. phagocytophilum were classified as "positive"(n=100), the following 100 negative dogs served as control group. In both groups, physical examination and laboratory parameters were compared. 22 (4.9%) dogs had antibodies to B. burgdorferi sl, 78 (19.4%) to A. phagocytophilum, nine (2.0%) to both agents. Bernese Mountain Dogs had significantly more often antibodies against B. burgdorferi sl. Negative dogs were more often diagnosed as "healthy" compared to A. phagocytophilum antibody-positives that showed more often elevated body temperature and poor general condition; beyond that, there were no differences in clinical and laboratory abnormalities between both groups. Although dogs tested negative were more often considered healthy, there were no differences in parameters considered "specific" for both infections between dogs with and without antibodies. Hence, tests detecting antibodies against both agents are not able to detect animals with the clinical disease.     

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.     

Exposure to infectious agents in dogs in remote coastal British Columbia: Possible sentinels of diseases in wildlife and humans

Ranked among the top threats to conservation worldwide, infectious disease is of particular concern for wild canids because domestic dogs (Canis familiaris) may serve as sources and reservoirs of infection. On British Columbia's largely undeveloped but rapidly changing central and north coasts, little is known about diseases in wolves (Canis lupus) or other wildlife. However, several threats exist for transfer of diseases among unvaccinated dogs and wolves. To gain baseline data on infectious agents in this area, including those with zoonotic potential, we collected blood and stool samples from 107 dogs in 5 remote communities in May and September 2007. Serology revealed that the dogs had been exposed to canine parvovirus, canine distemper virus, Bordetella bronchiseptica, canine respiratory coronavirus, and Leptospira interrogans. No dogs showed evidence of exposure to Ehrlichia canis, Anaplasma phagocytophilum, Borrelia burgdorferi, Dirofilaria immitis, or Cryptococcus gattii. Of 75 stool samples, 31 contained at least 1 parasitic infection, including Taeniid tapeworms, the nematodes Toxocara canis and Toxascaris leonina, and the protozoans Isospora sp., Giardia sp., Cryptosporidium sp., and Sarcocystis sp. This work provides a sound baseline for future monitoring of infectious agents that could affect dogs, sympatric wild canids, other wildlife, and humans.     

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.     

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.     

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.     

Molecular evidence of vector-borne pathogens coinfecting dogs from Poland

Ticks of the genus Ixodes are vectors for many pathogens, including Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp., and may also serve as vectors for Bartonella spp. However, the role of ticks in Bartonella transmission requires additional studies. The aim of this study was to investigate whether coinfection with two or more vector-borne pathogens can occur in the following three groups of dogs: I - dogs with suspected borreliosis (N = 92), II - dogs considered healthy (N = 100), and III - dogs with diagnosed babesiosis (N = 50). Polymerase chain reactions were performed to detect DNA of Anaplasma phagocytophilum, Rickettsia spp. and Bartonella spp. in the blood of dogs. In dogs of Group I, the DNA of both A. phagocytophilum and Bartonella sp. was detected (14% and 1%, respectively). In eight dogs, coinfection was indicated: A. phagocytophilum or Bartonella sp. with B. burgdorferi s.l. (the presence of antibodies against and/or DNA B. burgdorferi s.l.). In the case of five dogs positive for A. phagocytophilum DNA, no coinfection with B. burgdorferi s.l. was shown. In Group II, the DNA of A. phagocytophilum was detected in four dogs. In Group III, no pathogenic agents possibly transmitted by ticks were confirmed. No DNA of R. helvetica was detected in any of the groups studied.