Overview Of Anaplasmosis In Humans And Animals

Human behaviors such as migratory settlement, urbanization, movement across areas and travel have the ability to potentiate the exposure of new host reservoirs. Of highest importance is the rate and intensity of physical nearness between humans and animals. For instance, the Kenyan semi-arid and arid areas have often reported a high level of human wildlife interaction resulting in conflict which is as a result of encroachment into the wildlife ecosystem, game meat cultural practices and shared grazing fields among others.

This interaction could potentiate cross-species transmission and spill-over of diseases into new hosts from a reservoir species presenting local epidemics or global, pandemic emergence. While these diseases target humans as their main hosts, they are often in dormant circulation between wildlife animal reservoirs and tick vectors hence the need to focus on wildlife. Seventy-five percent of emerging infectious diseases (EIDs) according to Taylor et al., (2001) are considered of animal origin. Thus, a One Health approach at the human-animal-ecosystem interface is needed for effective investigation, prevention and control of any emerging zoonotic disease.

Anaplasmosis

Anaplasmosis is a tick-borne disease caused by obligate intracellular α-proteobacteria belonging to the genera Ehrlichia. Ehrlichia canis, was first recognized as a distinct clinical entity in Algeria in 1935. It has since been acknowledged worldwide as an important infectious disease of dogs and other canids with a higher frequency in tropical and sub-tropical regions.

Taxonomic classification

Anaplasma hemoparasites are in the family Anaplasmataceae, order of Rickettsiales, class Alphaproteobacteria and genus Anaplasma. Current improvements in molecular technologies have immensely advanced the potential to carry out genetic analyses. Based on the recent genetic analyses of 16S rRNA genes, groESL and surface protein genes reclassification of Anaplasma was done by Dulmer in 2001. This resulted in the inclusion of AP in to the genus which initially only included pathogens which were host specific for ruminants such as Anaplasma marginale and separating the Ehrlichia species from genus Anaplasma.

Morphology

AP is a small Gram-negative bacterium measuring up to 1.3um in size. In shape, it is pleomorphic coccoid to ellipsoidal and is enveloped by two membranes similar to most of the members of the family. According to Dumler et al., (2001) it often occurs in morulae usually 1.5 to 2.5 lm in diameter which are tightly packed formulations and in hematopoietic cells in domestic animals. These cells include myeloid cells, red blood cells and monocytes. Mitochondria do not come in close proximity to the morulae. There are two types of the bacterial cells; dense and reticulate, both present in the same vacuole and undergo binary fusion. They have no capsule layer but with an irregular periplasmic space created by the abundant outer membrane that invaginates into the bacterial protoplasm. Major antigenic membrane proteins, p44 and msp2, both of which are approximately 44-kDa in size are on the outer membrane surface of AP which are responsible for its virulence. Gnomically, AP has only one circular genome composed of 1471282 base pairs, composing 1264 protein genes with no known plasmids. Fine DNA strands and ribosomes are distinctly seen within the bacteria.

Host range

AP has a wide host range. Infections in animals are mainly found in domestic animals such as horses Dzięgiel et al., (2013), dogs Carrade et al., (2009) and cats. It has also been reported in livestock such as sheep, cattle, goats Woldehiwet (2010), dromedaries camels and llama. A number of wild animals especially wild ruminants have been reported as reservoirs in the USA.

Evidence by Woldehiwet (2010) reports subclinical persistent infections in deer. Baboons in Zambia have been reported to have strains of AP by Nakayima et al., (2014). In Europe, wild boars, red foxes and small mammals’ reservoir capacity has been vaguely established. Members of the Ixodes persulcatus transmit AP and maintenance is through its circulation between tick and small mammalian hosts such as the white-footed mouse (Peromyscus leucopus) which is a well described reservoir species in North America by Telford et al., (1996). Raccoon (Procyon lotor), and the gray squirrel (Sciurus carolinensis) Carlyon et al., (2003) are also mentioned with humans being accidental hosts.

Disease in man

AP causes a disease in humans called Human Granulocytic Anaplasmosis (HGA). Humans are an accidental host. In USA and Europe, HGA has been shown to be the third most common tick-borne disease by Dumler, (2012) and an emerging infectious disease in Asia. Clinical manifestations of HGA include fever, chills, headache, malaise and myalgia. Bakken et al., (1994); Chen et al., (1994) report leukopenia, thrombocytopenia and elevated levels of C-reactive protein and hepatic transaminases as being the most distinguishing manifestations of HGA among the laboratory findings. Additionally, cardiac manifestation with anaemia has been reported and increased aspartate and alanine aminotransferase activity arising from mild-to-moderate hepatic injury. Hospitalization is possible; however, most patients have mild clinical signs and symptoms. The gravity of the disease from asymptomatic to death. Acute respiratory distress syndrome and septic shock-like syndromes have been reported as possible complications with less than half of patients having skin rash and gastrointestinal tract symptoms such as diarrhea.

Disease in animals

Domestic animals have been reported to have clinical presenting signs of granulocytic anaplasmosis. In dogs, the most predominant signs reported by Mazepa et al., (2010) include fever, lethargy and inappetence. Kitaa, (2014) reported lymphadenomegally, congestion of mucous membranes, a pounding heart, pale mucous membranes, harsh lung sounds and vomiting in dogs. Feline granulocytic anaplasmosis is less common but Billeter, (2007) has reported of its recent increase in Europe and the United States. Haemorrhagic signs such as epistaxis, joint pain, lameness, lymphadenomegally in some cats, weight loss, a tender abdomen, conjunctivitis, myalgia, neck rigidity, neurological signs such as tremors, vomiting, pharyngitis, polydipsia, gingivitis or periodontitis among other as some of the presenting signs. In horses, the disease is equine granulocytic anaplasmosis and the infected show clinical signs of fever lasting up to 10 days one to three weeks postexposure. Most of the signs are similar to the other animals except limb oedema, orchitis, muscle stiffness, petechiation and icterus. Death may also be seen. Kuttler (1984) reported the presence of AP in cattle as a disease that produces progressive anemia and icterus. Other signs are high fever, coughs, miscarriages, decreased milk production and loss of appetite.

Other ruminants more or less have the same presenting signs including the wild ruminants which are considered as the reservoirs of AP alongside non-human primates. Rhesus macaques and baboons experimentally infected with AP had fever and anemia in one study, and fever, lethargy, anemia, thrombocytopenia and neutropenia in another.

Transmission

AP is spread by bites of infected Ixodes ticks. They belong to the family Ixodidae with a number of genera including ixodes which are hard tick as they have the scutum. The species include Ixodes spinipalpis, Ixodes persulcatus, Ixodes ovatus, Ixodes ricinus, Ixodes hexagonus, Ixodes scapularis, Ixodes trianguliceps and Ixodes Pacificus.

However, I. spinipalpis, I. persulcatus, and I. ovatus are the species in which AP DNA has been detected in the Asia. I. scapularis and I. pacificus Stuen et al., (2002) are the main vectors in United States, while Europe the main exophilic tick vector is I. ricinus. In rodents, I. trianguliceps has been implicated as the main vector in transmission. Felek et al., (2004) reported that naturally infected ticks can transmit AP to naïve mammals. The maintenance of the infection is highly dependent on the vertebrate host. AP is trans-stadially transmitted by these vector ticks as transovarial transmission has not been confirmed.

Ixodid ticks lodge on the skin using their mouth parts which lacerate the skin and takes a blood meal through their hypostome. They are then anchored on the host by secretion of proteins and lipids from specific cells in type II and III salivary granular acini. This allows them long feeding periods as a layer of cement is formed around the hypostome.

When feeding from an infected host, the tick acquires AP through the bloodmeal. The bacterium is maintained from the larval stage to adult stages of metamorphosis and is transmitted to mammals during the next blood meal as saliva from salivary glands and midgut is containing AP is deposited.

Replication

AP accesses the bloodstream after the tick bite reaching the intracellular environment. Entry and intracellular infection by AP require lipid rafts, which are signaling platforms. This is where AP concentrates. For instance, phosphorylation of protein tyrosine is induced once AP has bound resulting in internalization and infection. It then multiplies strictly within the cell membrane-derived vacuole in the cytoplasm of the vertebrate host’s cell colonizing it. As bacteria divide and proliferate, the inclusions expand to occupy most of the cytoplasm of infected cells. These inclusions appear to evolve over time as the AP infection cycle progresses. Linkage between AP and endothelial cells has been shown as it has been suspected that the initial step after transmission is AP infecting the endothelium before the granulocyte infection. Once bound, AP stimulates the release of a chemokine, Interleukin 8(IL-8) which attracts neutrophils to the site of infection probing microbial infection. In circulating mature neutrophils is where AP is maintained instead of peripheral tissues, including hematopoietic tissue although bone marrow progenitors can also be infected. Normally, neutrophils phagocytise bacteria using phagosomes, lysosomal hydrolytic enzymes and sequestering vital nutrients. However, AP avoids lysis by interfering with vesicular trafficking.To enter the blood circulation, now tightly packed AP in the inclusions squeeze within the limited intravacuolar space while maintaining the plasticity of the infected granulocytes into the capillary circulation.

Strain variation and diagnostic techniques

According to Wuritu et al., (2009), there appears to be an existence of serological cross-reactivity in strains of AP with the most common constitutively produced antigens being 42- to 49-kDa proteins, which are expressed on the bacterial outer membrane.

The proteins are encoded by the p44 gene family which is the main antigenic protein of AP and is highly polymorphic. There is a minor degree of variation in the nucleotide sequences of the 16S rRNA, groESL, gltA, ank, and msp2 genes, with the exception of some ank sequences from infected German ticks that are different from other ank sequences of human and animal strains. Strains clearly differ; not all appear to be capable of infecting humans or mice Massung et al., (2003) or to cause persistent infections. There is also a difference in the host infectivity of AP. For instance, Foley et al., (2009) reported that AP strains from wild rodents were reported to differ in horse infectivity and Stannard et al., (1969) on strains infectious to equine being non-infectious to ruminants.

Direct microscopy

This is the simplest technique for detection and it involves observing a blood smear stained with Giemsa or Wright through a microscope from animals clinically showing the disease acutely. For a positive smear, what is observed is a cluster of bacteria called morulae within cytoplasmic vacuoles in peripheral blood neutrophils.

Immunohistochemistry

This has seldom been used to examine blood and tissues to detect specific antibodies which might unfortunately not be readily available commercially. There is scarce literature in description of this technique.

Serology

This is often used to detect the presence of antibodies especially for animals not showing the clinical disease. For the epidemiological studies of Anaplasma, a number of serological tests have been employed despite the fact that they do not differentiate varied Anaplasma species resulting often in cross-reactivity among the species. Competitive enzyme-linked immunosorbent assay (cELISA) is mostly used to detect anti-major surface protein 5 (anti-MSP5) of Anaplasma species as it reliably establishes a carrier state.

Indirect fluorescent antibody (IFA) is another technique commonly used as it is highly sensitive and specific. However, the fact that there may be a reaction with other autoimmune antibodies renders it faulty.

Molecular diagnosis

This diagnostic technique focuses on amplifying nucleic acids specific for AP and confirmation done by sequence analysis which allows for identifying genus and species of these microorganisms. These methods include conventional PCR which targets msp2 gene of AP to identify animals that have been infected naturally and real-time PCR (qPCR) which was recreated to detect more than one species. Loop-mediated isothermal amplification (LAMP) assay is for rapid detection of AP using primers specific to 16S rRNA gene of this organism as it amplifies novel nucleic acid. It is cost-effective and a simple detection technique.

Molecular diagnosis is the method of choice as its sensitivity and specificity are high. Genetic characterization by use of DNA markers and comparing it with the gene bank allows for one to know the genetic variation present as well as to identify the phylogenetic and molecular structure of the pathogen.