Anaplasma phagocytophilum, the contributing agent of human granulocytic anaplasmosis and tick-borne fever, is the recently renamed obligate intracellular bacterium following the restructuring of the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiaceae. It causes disease in humans, horses, dogs, and ruminants. This name was given replacing three species of granulocytic bacteria, Ehrlichia phagocytophila, Ehrlichia equi and the agent of human granulocytic ehrlichiosis which were previously classified as separate species because of various hosts and relatively remote geographic locations. The new taxonomic assignment creates a single species but allows for biological and clinical heterogeneity (Dumler et al. 2003). The prototype of Anaplasma phagocytophilum has been reorganized as a pathogen of domestic and free living ruminants in Europe for over 70 years. It was first described in 1940, 8 years after the first acknowledgment of TBF as a distinct tick transmitted disease in Europe.
Anaplasma phagocytophilum was initially considered to be a member of the genus Ehrlichia, together with the other major pathogen causing a similar human disease, E. chaffeensis, however recent analysis if DNA sequences particularly the 16S and groEL genes have led to the reclassification as an Anaplasma species (Goodman 2005). This analysis showed that there were only minor differences among Ehrlichia phagocytophila, Ehrlichia equi, and the human granulocytic ehrlichiosis (HGE) agent; the new classification creates a single species but allows for biological and clinical variations. The family Anaplasmataceae has been broadened to include four distinct genera Ehrlichia, Anaplasma, Wolbachia and Neorickettsia. All of the members in this family are small Gram-negative pleomorphic cocci that are obligate intracellular bacteria and replicate in membrane bound vacuoles (Rikihisa 2006). The bacterium in the family Anaplasmataceae is not contagious but vector borne.
Epidemiology and geographical distribution
A variety of strains of Anaplasma phagocytophilum have been shown to cause disease in a wide range of mammalian hosts worldwide including the United States, Asia and numerous European countries. The life cycle of A. phagocytophilum involves a complex relationship between its natural hosts or reservoirs and its tick vectors that develop through multiple life stages and may transmit an infection to animals and humans. This requires that the bacterium has the ability to adapt to these multiple environments and environmental pressures (Lin et al. 2004). In the United States, infections with HGA have shown up in areas such as the Northeast and upper Midwestern states, with California having the most case reports for equine granulocytic anaplasmosis. Tick-transmitted diseases are a major threat to human and animals around the world. Humans spend majority of the summer months pursing outdoor activities which has increased their potential exposure to pathogenic bacteria that are present in nonvertebrate bloodsucking enzootic hosts during a portion of their life cycle (Bakken and Dumler 2006).
Equine granulocytic ehrlichiosis, which was not previously recognized as Equine granulocytic ehrlichiosis, was first reported as a disease of horses in California and was later established as an equine disease in other parts of the USA and Europe. Canine granulocytic anaplasmosis (CGA) was also first recognized in the USA before its more recent introduction into Europe. Until the recent discovery of human granulocytic anaplasmosis, which was previously human granulocytic ehrlichiosis in the US, it was thought that this disease only spread between domestic animals and free-living reservoirs. The recognition that HGA is caused by an agent closely related to the causative agent of TBF in ruminants, and EGE in horses has created a renewed interest resulting in more information on their molecular biology and pathology (Woldehiwet 2009).
Pathogenesis and clinical presentation
The tick-borne rickettsial disease, human granulocytic anaplasmosis, was first reported in the United States in the 1990’s as a disease that is a threat to human life. Anaplasma phagocytophilum had previously been known as a ruminant pathogen in Europe. The first case of HGA was reported in 1990 in a Wisconsin patient who died from a severe febrile illness two weeks after being bitten by a tick (Dumler at al. 2005). HGA has become an increasingly important disease to expect in the areas of the United States where Ixodes ticks are known to bite humans. There have been a total of 3637 HGA cases reported in the USA from 2003 to 2008, with 2007 having the most case reports at 834 (Thomas et al. 2009).
The most common symptoms reported include: muscle pain, general pain and discomfort, fever and few cases of nausea and respiratory distress. In tick-endemic areas symptomatic infections occur but infections vary from mild, self limited fever to death. Infections severe enough for hospitalization occurs in half of the symptomatic patients and are attributed to factors such as: older age, higher neutrophil counts, anemia, the presence of morulae in leukocytes, or underlying immune suppression. Roughly 5-7% of patients infected with HGA require intensive care. However, at least 7 deaths have been reported that resulted from delayed diagnosis and treatment (Dumler at al. 2005). Studies have shown that for the most part HGA is considered to be a mild or asymptomatic infection and can be treated with in 1 to 2 weeks with antibiotics. A. phagocytophilum infections in Europe are relatively different from the infections in the United States. Human HGA infections are less frequent in Europe; however, infections in livestock are much more common and cause a tremendous burden on the livestock industry.
Ticks are blooding-feeding ectoparasites of animals and humans. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodidae and are dispersed worldwide from Artic to tropic (de la Fuente at al. 2007). Ixodes ticks are the recognized vectors of the tick transmitted bacterium Anaplasma phagocytophilum. In the United States Ixodes scapularis is the known vector for HGA in the eastern and midwestern regions, whereas Ixodes pacificus is the known vector for the Pacific coast. Approximately 10-50% of Ixodes scapularis ticks are infected with A. phagocytophilum in that U.S. Tick infection is established after an infectious blood meal on transiently and persistently infected reservoir hosts. Transovarial transmission does not occur; therefore descendants from infected female ticks are not infected and must obtain the infectious agent in a successive blood meal. Adult Ixodes spp. ticks in the northern USA are active during from March through June and October through December. The nymphs have the highest active rate from Mat to September, while larval activity is more prominent from June to October (Thomas et al. 2009).
The host range of Anaplasma phagocytophilum appears to vary according to geographical regions; therefore, it has the possibility to infect numerous hosts. The occurrence and severity of the disease in a particular host also appears to vary from one region of the world to the other. This variation is largely dictated by the strain or variant of A. phagocytophilum infecting the reservoir hosts, the secondary and reservoir hosts to which it has adapted and the capacity of the vectors present in a particular region (Woldehiwet 2009). Previously A. phagocytophilum was thought to be maintained in a tick-ruminant cycle only. Recent studies done on the various strains of A. phagocytophilum have shown that in the USA, the main reservoirs of HGA are the vertebrate hosts of the black-legged tick I. scapularis, the most prominent one being the white footed mouse (Peromyscus leucopus), but the host list also includes the white-tailed deer (Odocoileus virginianus), grey squirrels (Sciurus carolinensis), and the raccoon (Procyon lotor) (Woldehiwet 2009).
Anaplasma phagocytophilum persists within the secretory salivary acini of tick salivary glands. Tick feeding stimulates the replication and migration of the bacteria from the salivary glands to the mammalian host. Studies suggest that transmission of A. phagocytophilum occurs between 24 and 48 h after tick attachment (Sukumaran at al. 2006). A. phagocytophilum’s strict intracellular location provides a mechanism for evading host defenses and also promotes chemotactic mechanisms (IL-8) that assist the attraction of neutrophils to the tick bit site (Granquist at al. 2010). Anaplasma phagocytophilum is a small intracellular bacterium with a gram-negative cell wall; it is one of only four bacteria known to survive within human neutrophils. Arthropod-borne intracellular organisms that parasitize the cells of mammalian hosts must be able to manipulate a diversity of host cells to maintain their own growth and life cycle (Nelson et al. 2008). Neutrophils are thought to be incompatible host cells for intracellular bacteria because they are short-lived and are the principal defense cells.
One of the main functions of neutrophils is to ingest and kill invading microorganisms; this is a fundamental part of natural immunity. An infection with A. phagocytophilum causes major changes in neutrophil functions, which ultimately results in clinical disease. Apoptosis of infected cells is one of the important natural immune responses against intracellular pathogens, including viruses, bacteria, and parasites. Neutrophils typically undergo spontaneous apoptosis within 6-12 h after release into the peripheral blood from the bone marrow, an important process in the maintenance of homeostatic levels of neutrophils and in the resolution of inflammatory responses. A. phagocytophilum infection inhibits spontaneous and induced apoptosis of isolated peripheral blood human neutrophils for up to 48 h and of neutrophils in peripheral blood leukocyte cultures for up to 96 h as determined by morphological observation (Niu et al. 2010). Following the infection with the bacterium, A. phagocytophilum divides until cell lysis or bacteria are discharged to infect other cells.
Agent and pathogen development cycle
Arthropod-borne infectious diseases are a major threat to both domestic and wild animals as well as humans. Humans that have been infected with HGA show clinical signs such as: fever, headache, myalgia, malaise, leucopenia, and thrombocytopenia. TBF positive ruminants show clinical signs varying from indictable illness to severe disease associated with opportunistic infections, hemorrhage, and abortions. Equine and canine infections are characterized by fever, depression, anorexia, leucopenia, and thrombocytopenia. The clinical manifestations HGA infections range from mild to fatal, the infection is sometimes associated with other infections. HGA for the most part is considered to be a mild infection and can be treated within a couple of weeks with antibiotic treatment. The estimated HGA case fatality rate is low (0.5 % to 1%); however, it may be difficult to identify patients who are likely to develop serious or fatal disease. Thus, prompt institution of active antibiotic therapy advocates for all patients who have confirmed HGA are symptomatic (Bakken and Dumler 2006). The severity of the infection is influenced by several factors, such as variants of A. phagocytophilum involved, other pathogens immune status and condition of the host, and factors such as climate and management (Stuen 2007). Even if treated with antibiotics humans can have unfavorable outcomes from the infection such as persistent fevers. Infections in ruminants and rodents can be persistent as well and blood taken from these animals by a feeding tick can infect naïve animals.
Clinical and Laboratory diagnosis
In clinical infections with human granulocytic anaplasmosis, membrane bound A. phagocytophilum colonies, called morula, appear in peripheral blood neutrophils. A. phagocytophilum diagnosis can be confirmed in the early infection stages by blood smear examination and PCR analysis and in the late infection stages by serologic testing. PCR amplification of A. phagocytophilum DNA from acute-phase blood or isolation of A. phagocytophilum in HL-60 promyelocytic leukemia cell cultures inoculated with acute-phase blood can confirm the diagnosis. Test done with blood samples need to be held and ran before the patient begins antibiotic treatment since treatment will rapidly reduce the detectable quantities of infected cells or bacterial DNA (Bakken and Dumler 2006). Little is known about how the infection spreads from the initial tick bite site, what cells or tissues are involved, what causes this signs of illness, and when severe cases exist, how long the tissue damage will persist (Goodman 2005).
Propagation in cell culture
In cell culture Anaplasma phagocytophilum can grow and infect two tick cell lines IDE8 and ISE6 as well as HL-60 cells. HL-60 is a promyelocytic leukemia-derived cell line, that retains many characteristics of the human leukocyte progenitors that A. phagocytophilum is known to infect, including the potential to undergo partial differentiation into morphologically mature neutrophils. HL-60 cells unlike neutrophils; lack formyl-Met_Leu-Phe and interleukin (IL-8)-induced chemotaxis, and IL-8 is upregulated during A. phagocytophilum infection and is an important neutrophil recruitment chemokine for A. phagocytophilum survival and they do not rapidly undergo apoptosis like neutrophils (Lee et al. 2008). Although there are differences HL-60 cells have been very valuable in studying the molecular and cellular biology of A. phagocytophilum infections. A. phagocytophilum is able to create distinct infection phenotypes and growth kinetics in these cell lines, which suggest that along with its broad host range, the organism has the ability to shift its gene expression to adapt to each host (Nelson et al. 2008).
A. phagocytophilum has a high degree of diversity in clinical disease that exists among the different strains of the bacterium. The organism is able to adapt to different environments due to gene duplications, these duplications underlie the diversification of the genes, with the results often being the development of novel gene function or pseudogenes that may help the organism adapt to new environments (Lin et al. 2004). As shown from the sequencing of amplified fragments of the 16s rRNA gene, the TBF variants differ from the EGA/HGA variants in three positions (Woldehiwet 2010). A. phagocytophilum has 44-kDa immunodominant major surface proteins that are encoded by the p44/msp2 multigene family (Wuritu et al. 2009). The Omp-1/P44/Msp2 superfamily is the most studied outer membrane protein family of A. phagocytophilum, the genome has 121 genes belonging to this superfamily: one msp2, two msp2 homolos, one msp4, 113 p44, and three comp-1. P44 and Msp2 proteins are homologous yet distinct groups of proteins (Lin et al. 2004). The transcription of the various p44 genes gives way to the antigenic variation of A. phagocytophilum. P44s play an important role in the pathogenesis of A. phagocytophilum in its mammalian host. The diversity of these genes and the surface protein they encode for may reflect the differences among the geographic regions and the host specificity (Lin et al. 2004).
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