Equine Granulocytic Anaplasmosis Research Articles

Strains of Anaplasma phagocytophilum from horses in Ohio are related to isolates from humans in the northeastern USA.

The tick-borne obligatory intracellular bacterium Anaplasma phagocytophilum infects granulocytes of humans as well as domesticated and wild animals, causing a febrile disease collectively called granulocytic anaplasmosis. The host species specificity and zoonotic potential of A. phagocytophilum strains remain unclear. In the USA, human granulocytic anaplasmosis (HGA) is primarily reported in Wisconsin and Minnesota, the northeastern coastal states, and northern California. In horses, A. phagocytophilum causes equine granulocytic anaplasmosis (EGA). Although EGA has been reported in the same US regions as HGA, horses with a definitive diagnosis of EGA have not been reported in Ohio, where the incidence of HGA is low. In this study, samples from archived blood smears or whole blood from three horses from three different counties in Ohio that presented in 2018, 2020, and 2021 with clinical signs and laboratory findings consistent with EGA were evaluated by PCR and sequencing. Sequences of ankA (encoding ankyrin A) of the three A. phagocytophilum strains were identical and related to strains isolated from humans in the northeastern USA yet distinct from those in California or Europe based on phylogenetic analysis. Each Ohio strain contains unique A. phagocytophilum p44 genes encoding the immunodominant major outer membrane proteins, underscoring the antigenic diversity of A. phagocytophilum strains that cause EGA in Ohio. From 2018 to 2023, 14 additional cases of EGA were identified by serodiagnosis in Ohio and nearby locations in Pennsylvania, pointing to a heretofore unrecognized prevalence of EGA and the risk of transmission of anaplasmosis to humans and animals in this region.IMPORTANCEThe tick-borne obligatory intracellular bacterium Anaplasma phagocytophilum infects humans as well as domesticated and wild animals, causing a febrile disease collectively called granulocytic anaplasmosis. The epidemiology and the host species specificity and zoonotic potential of A. phagocytophilum strains remain unclear. In this study, ankA (encoding ankyrin A) and p44 gene sequences of A. phagocytophilum were determined in clinical specimens from horses in Ohio and compared with those found in A. phagocytophilum strains from various hosts and geographic regions. With increasing numbers of seropositive horses, the study points out the unrecognized prevalence and uncharacterized strains of A. phagocytophilum infection in horses and the importance of A. phagocytophilum molecular testing for the prevention of equine and human granulocytic anaplasmosis.

Phylogenetic identification of Anaplasma phagocytophilum in horses in Baghdad, Iraq

This study aimed to detect Anaplasma phagocytophilum in horses through hematological and molecular tests. The 16S rRNA gene of the Anaplasma phagocytophilum parasite was amplified by polymerase chain reaction (PCR), then sequenced, and subjected to phylogenetic analysis to explore "Equine Granulocytic Anaplasmosis" (EGA) infection in three important gathering race horses areas in Baghdad governorate, Iraq. Blood samples were obtained from 160 horses of varying ages, three breeds, and both sexes, between January and December 2021. Prevalence and risk variables for anaplasmosis were analyzed using statistical odds ratio and chi-square tests. Results demonstrated that clinical anaplasmosis symptoms comprised jaundice, weight loss, paleness of mucus membrane with petechial hemorrhage in the third elides, and edema in extremities; There was no tick infestation. The hematological test did not significantly reveal decreases in red and white blood cells and platelet count. Microscopically found 11 from 160 smears (6.88%) had morulae within granulocytes, PCR results of Anaplasma spp primers was 32 positive amplicons (20%), and molecular sequencing results of “16S ribosomal RNA genes” confirmed 21 horses (13.13%) infected by Anaplasma phagocytophilum for the first time in Iraq horses. The results of the phylogenetic analysis revealed compatibility values similarity 98.81-99.76% with worldwide isolates. Mares occurred not significantly riskier; also age and breed were not illustrated risks of any group. This study is the first molecular detection of Anaplasma phagocytophilum in racehorses reared in Baghdad in Iraq. The outcomes of this study provide genetic data for early identification of Anaplasma phagocytophilum infection, treatment, and management of the illness in Iraq horses, as well as monitoring its transmission to the human population.

Anaplasma phagocytophilum in Horses - Evaluation of Proinflammatory Biomarkers

ABSTRACTBackground: Anaplasma phagocytophilum is an obligate intracellular pathogen transmitted by the ticks that cause equine granulocytic anaplasmosis (EGA). This pathogen is infects predominantly blood cells, principally granulocytes and especially neutrophils. A. phagocytophilum causes an acute febrile disease in horses accompanying with lethargy, loss of appetite, lameness and hemorrhages. In horses, this disease should be considered in all acute symptoms accompanied by thrombocytopenia and leukopenia identified by hematological test performed. Tick-borne pathogens have become increasingly threatening for both animals and also public health since ticks mostly carry numerous well-documented and undocumented pathogens, and the geographical range of ticks has expanded in the recent years. This research has aimed to evaluate the impact of A. phagocytophilum infection on some oxidative/nitrosative stress parameters, antioxidant enzyme activities, proinflammatory biomarkers and trace element levels in horses.Materials, Methods & Results: The present study has been carried out using blood samples collected from 93 horses aged 1-year and older. The blood samples were centrifuged and sera were separated. Serum samples stored in the freezer (-20°C) until the day of analysis. The DNA was extracted from blood and analysed by nested-PCR technique targeting 16S rRNA gene of A. phagocytophilum and then positive PCR products were sequenced. A. phagocytophilum was 6 horses (6.4%) showed positive nested-PCR results. An infected group comprised of 6 positive horses according to PCR analysis results also 6 healthy horses as control were selected. Serum SOD (Horse Superoxide Dismutase(Cu-Zn)) ELISA Kit, MPO (ELISA Assay Kit Horse Myeloperoxidase) and GPx (Horse glutathione peroxidase 1 ELISA Kit Assay), IL1 (Horse Interleukin 1 Beta ELISA Kit), IL6 (Horse Interleukin 6 ELISA Kit), TNF α (Horse Tumor Necrosis Factor Alpha ELISA Kit) and IL18 (Horse Interleukin 18 (IL18) ELISA Kit) levels were determined by ELISA reader. Serum TAS ,TOS Glucose-6-phosphate dehydrogenase (G6PD) levels were determined using colorimetric kit method. The determination of peroxynitrite was performed using spectrophotometry as described by Vanuffelen. The levels of Fe, Zn, Se, Cu, Mn, Ar, Cr, Co, Cd Ni and Pb elements were analyzed by ICP-OES. Total oxidant status (TOS) and peroxynitrite levels of the positive group infected with A. phagocytophilum were found to be higher compared with the control group (P < 0.05, respectively). Whereas, total antioxidant status (TAS), glutathione peroxidase (GPx) ,superoxide dismutase (SOD), G6PD and myeloperoxidase (MPO) levels were detected to be statistically significantly higher than the control group (P < 0.05, respectively). Cu, Mn, Se and Zn levels were also found to be lower in the infected group (P < 0.05, respectively). IL1, IL6, TNFα and IL18 from proinflammatory markers were elevated in the A. phagocytophilum infected group (P < 0.05, respectively).Discussion: The evaluation of the proinflammatory biomarkers with respect to host-parasite interactions has been suggested as a beneficial clinical tool for determination of the infection severity and diagnosis of the disease. As a conclusion, increased oxidative stress and high levels of some proinflammatory biomarkers assessed by the multibiomarker analysis carried out for the infection in the horses caused by Anaplasma phagocytophilum will provide a contribution to diagnosis, treatment and clarification of the pathogenesis.Keywords: anaplasmosis, EGA, tick-borne pathogens, pro-inflammatory cytokine, antioksidant, oxidative/nitrosative stress, equine.

Equine anaplasmosis and equine piroplasmosis in Germany, Austria and Switzerland - previously anecdotal, now relevant?

Equine granulocytic anaplasmosis (EGA) and equine piroplasmosis (EP) are triggered by tick-borne pathogens - the intracellular bacterium Anaplasma phagocytophilum and the intracellular protozoa Babesia caballi and Theileria equi. These pathogens attack cells in the blood stream and cause similar clinical symptoms and changes in laboratory values. Although the treatment principles are naturally different, similarities in prophylaxis exists due to the transmission route. Tick transmitted pathogens can play a greater role in equine medicine in the future due to various factors, such as the tendency of relevant tick species to spread, but also the increasing import and travel activities of and with pets (both in the context of sporting events and as a leisure activity). While EGA is endemic in Central Europe, EP is a sporadic disease in Switzerland, Austria and Germany. However, EP must be viewed as underdiagnosed, as horses persistently infected with T. equi are also repeatedly detected in Central Europe. These diseases should be considered in horses with a fever and corresponding laboratory changes. Available diagnostic tests are direct pathogen detection by blood smear or PCR, and, indirect antibody detection, which is considered to be highly sensitive and (as a competitive ELISA) also very specific. Acute infections can be detected with PCR, serology is more suitable for chronic infections. A pathogen-free condition after treatment can be demonstrated with decreasing antibody titers in combination with repeated PCR tests. In addition, clinically healthy horses infected with T. equi should be identified by antibody detection and appropriate preventative transmission measures must be initiated. The prophylaxis of tick bites in horses is difficult due to the high exposure, and long-term tick bite prevention can hardly be guaranteed. Monitoring of tick activity and strict measures to prevent the spread of the pathogen within the tick population are therefore of great importance.

Equine Lyme disease and equine granulocytic anaplasmosis Part 1 – Potential risk factors (owner survey)

Equine Lyme disease and equine granulocytic anaplasmosis Part 1 – Potential risk factors (owner survey)

Equine Lyme borreliosis and equine granulocytic anaplasmosis Part 2 – Clinical relevance (Vet survey)

Equine Lyme borreliosis and equine granulocytic anaplasmosis Part 2 – Clinical relevance (Vet survey)

Hematology and serology analysis of healthy and Anaplasma phagocytophilum infected equids in Minas Gerais, Brazil

Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes equine granulocytic anaplasmosis (EGA), that is characterized by fever, anorexia, depression, limb oedema. Hematological and serological alterations are important findings during the disease. We analyzed whole blood and serum from 37 horses and mules from three locations in Minas Gerais, Brazil, for total blood cell count, leucocytes, platelets, renal and hepatic profiles. Equids infected by A. phagocytophilum had differences in blood cell count and renal and hepatic profiles. As a zoonosis, the study of equine A. phagocytophilum infection in areas with high population density and in breeding farms, is important to understand the disease dynamics and avoid human cases of anaplasmosis.

Türkiye’nin Muş İlinde Atlarda Anaplasma phagocytophilum’un İlk Moleküler Tespiti ve Filogenetik Analizi

Objective: Equine granulocytic anaplasmosis (EGA) caused by Anaplasma phagocytophilum is a disease that generally develop characteristic high fever and a number of changes in blood such as thrombocytopenia. The main transmission of A. phagocytophilum is hard ticks through intrastadially. The aim of this study was to determine the presence of A. phagocytophilum in horses in Muş province of Turkey by using molecular methods. Methods: In the present survey, blood samples were collected in Muş province from 93 horse. Commercial ELISA kit (IgG) and nested-PCR were performed. The DNA was extracted from blood and analysed by nested-PCR technique targeting 16S rRNA gene of A. phagocytophilum and then positive PCR products were sequenced. Results: The seroprevalence against A. phagocytophilum was 8.6% and 6 horses (6.4%) showed positive nested-PCR results. The sequences had 99.5 and 100% homology with the BLAST published A. phagocytophilum amplicons. Conclusion: To the best of our knowledge, this is the first molecular data of A. phagocytophilum in horses in Muş province of Turkey. This study also reveals that A. phagocytophilum isolates isolated from a small region are closely related to A. phagocytophilum isolated from a wide variety of hosts.

A survey on equine tick-borne diseases: The molecular detection of Babesia ovis DNA in Turkish racehorses

Common vector-borne diseases of horses include equine piroplasmosis (EP) caused by Babesia caballi and Theileria equi, and equine granulocytic anaplasmosis (EGA) caused by Anaplasma phagocytophilum. Equine piroplasmosis leads to severe health issues in horses and restrictions on the movement of horses internationally. Anaplasma phagocytophilum causes an acute febrile illness in horses and is also of zoonotic importance. In the present study, blood samples were collected from 152 Turkish racehorses from three different provinces (İzmir, Gaziantep, and Konya) of Turkey to investigate the prevalence of EP and EGA. Standard and nested polymerase chain reactions were performed to identify equine piroplasms and A. phagocytophilum, respectively. PCR primers targeting Babesia spp. 18S rRNA, B. caballi BC48, T. equi EMA-1, and A. phagocytophilum 16S rRNA genes were used for molecular diagnosis. Following the cloning and subsequent sequencing of PCR-positive samples, a total of 15 (9.9%) horses were found to be infected with at least one pathogen. Theileria equi and A. phagocytophilum were found in 3.3% (5/152) and 6.6% (10/152) of the samples, respectively. Although B. caballi specimens were not detected in any of the samples, a positive signal was detected for the Babesia genus-specific 18S rRNA PCR. Subsequent sequencing of this signal revealed 100% identity to Babesia ovis. This is the first detection of B. ovis DNA in racehorses in Turkey to the best of our knowledge. Additionally, this study also reports the first molecular identification of A. phagocytophilum in Turkish racehorses. Based on this report, it is recommended that future epidemiological studies on horses also take B. ovis, a parasite usually found in sheep, into consideration and that further detailed studies be conducted to unravel the transmission pathways and potential clinical effects of B. ovis in horses.

147 Serological and molecular detection of Anaplasma in East Texas horses

Anaplasma phagocytophilum (AP) is a tick-borne bacteria that cycles between the tick, Ixodes scapularis and the hematopoietic cells of vertebrate hosts. AP is responsible for the disease equine granulocytic anaplasmosis, a generally transient infection characterized by fever, depression, inappetence, ventral edema petechiae, icterus, ataxia, recumbency, muscle stiffness, and, in severe cases, death. Current infection with AP is detected through PCR analysis of an animal's immune cell DNA, while serum antibody titers determine if a horse has previously been infected with AP. Other regions and countries have reported wide ranging prevalence of serum reactive horses, with 17 to 67% of apparently healthy horses being serum reactive and 8% of these being PCR positive. There were 130 clinically healthy horses aged from 1 to 29 years old included in this survey, 70 from Texas, and 11 from New Jersey, and 49 from New York. Horses from Texas were chosen based on location in the state and high potential for tick exposure, the latter 2 states were included as populations with known tick exposure. From each horse, blood was collected for serum and DNA. DNA was screened by nested PCR for the 23s rRNA gene of the Anaplasmataceae family. Samples positive for 23s were analyzed by conventional PCR for the msp2 gene of AP. Lastly, serum was analyzed by immunofluorescence antibody test to detect antibodies against AP. Seventeen horses were positive for 23s, with 4, 3, and 10 from Texas, New Jersey, and New York, respectively. Of these, 2 Texas horses were positive for msp2. Further, 107 horses tested positive for antibodies against AP, with 47, 11, and 49 from Texas, New Jersey, and New York, respectively. The observed 80% seroprevalence is similar to that reported from other countries and indicates high exposure to tick populations.

Equine Granulocytic Anaplasmosis, A Neglected Disease: Risk Factors Associated with Prevalence of Antibodies in Equines

Background: Anaplasma phagocytophilum, a tick-borne bacterium that causes granulocytic anaplasmosis, is a neglected pathogen in Brazil, and is diagnosed in several species of domestic and wild animals as well as in humans. Objective: This study aimed to investigate the prevalence of anti-A. phagocytophilum antibodies in Equidae from the state of Rio de Janeiro and to identify possible risk factors for infection. Materials and Methods: A total of 612 blood samples were collected from horses from 15 municipalities within the state. Moreover, an epidemiological questionnaire was administered to evaluate aspects related to seroreaction, taking into account the spatial distribution (properties, municipalities, and mesoregions), management practices, signs of disease, and the individual state of the animals. For the diagnosis, indirect immunofluorescence was performed. Results: In the present study, 124 (20.26%), out of a total of 612, animals with anti-A. phagocytophilum IgG antibodies at titers of 1:80 were detected. Multivariate logistic regression analysis showed that the presence of the infection at the property (P <0.0001) and the origin (P = 0.0095) of the horse were the true risk factors for infection in the state of Rio de Janeiro. Conclusion: This allows to infer that the bacterium is distributed in all mesoregions of the state of Rio de Janeiro and that animals from other states can introduce the infection and make a property a focus of disease; it can also be inferred that these properties are important in the maintenance of the disease and the permanence of bacteria circulating in horses. It is also noteworthy that this was the first identification of mules as hosts of A. phagocytophilum infection.

Serological and molecular detection of Anaplasma phagocytophilum in Thoroughbred horses from Chilean racecourses

Anaplasma phagocytophilum is the causative agent of equine granulocytic anaplasmosis (EGA). This study aimed to perform serological and molecular surveys of A. phagocytophilum in thoroughbred horses from racecourses in Chile. Additionally, hematological findings related to A. phagocytophilum molecular positivity were addressed, and phylogenetic analysis of selected positive samples was performed. Complete blood count and msp2 gene real-time PCR were performed in 457 thoroughbred horses from three racecourses located in three different cities of Chile (Santiago, Viña del Mar and Concepción). Sera from horses in two racecourses (Santiago and Vina del Mar) were tested by Indirect fluorescent antibody test (IFAT) to detect IgG antibodies against A. phagocytophilum. The occurrence of A. phagocytophilum by real-time PCR was 13.6 % (62/457, 95 % CI: 10.8–16.3 %), with the highest occurrence observed in Santiago (26.5 %), followed by Concepción (9%), and the lowest in Viña del Mar (5%). The overall frequency of IgG antibodies to A. phagocytophilum was 7.9 % (23/290, 95 % CI: 4.8–12.7 %), with 9.9 % in Santiago and 6.5 % in Viña del Mar. Only three animals from Santiago Racecourse were positive in both real-time PCR and serology. PCR-positive horses from Santiago racecourse presented significantly lower hemoglobin, mean corpuscular value (MCV), and mean corpuscular hemoglobin concentration (CHCM), and higher eosinophil counts. Phylogenetic analysis based on the msp2 gene showed that A. phagocytophilum sequences found in the present study were closely related with A. phagocytophilum sequences from the USA and Europe. Anaplasma phagocytophilum DNA is detected for the first time in Chile.

Transplacental infection of a foal with Anaplasma phagocytophilum

SummaryThis is the first report to document transplacental transmission of Anaplasma phagocytophillum in the horse. A 4‐year‐old late‐term pregnant mare presented for a recent onset of pyrexia due to equine granulocytic anaplasmosis (EGA). She was hospitalised for treatment with oxytetracycline and monitoring of high‐risk foaling due to significant thrombocytopenia. Parturition occurred overnight, and the foal was PCR positive for A. phagocytophilum at birth. The foal was slow to stand and nurse, with signs of neonatal encephalopathy and anaplasmosis (thrombocytopenia). Therapy with oxytetracycline resulted in complete clinical recovery of the mare and foal within 5 days. Congenital anaplasmosis should be considered in any foal delivered to a mare suffering from EGA during late‐term pregnancy and guide appropriate antimicrobial therapy.

Equine Granulocytic Anaplasmosis in Southern Sweden: Associations with coniferous forest, water bodies and landscape heterogeneity

Landscape characteristics influence both vector and host habitats affecting the spatial and temporal distribution of vector-borne diseases. Anaplasma phagocytophilum is one of the most widespread tick-borne diseases in Europe causing tick-borne fever (TBF) in domestic ruminants, and granulocytic anaplasmosis in humans, horses and companion animals. The aim of this study was to identify landscape factors associated with Equine Granulocytic Anaplasmosis (EGA) cases in a refined temporal and spatial analysis by combining land cover data and presence/absence disease data, using a geographical information system (GIS). This study is a retrospective analysis utilizing 1030 EGA diagnostic test results extracted from the National Veterinary Institute (SVA, Sweden). The association between EGA and different land cover types was analyzed with generalized linear models. To analyze the relation between landscape heterogeneity and EGA, we calculated the Gini-Simpson index. Our results showed a significant increase in the proportion positive EGA cases from 2002 to 2015 and marked differences in the seasonal within year distribution of EGA cases. The association with landscape configuration is shown by the positive relationship between A. phagocytophilum and coniferous forest, water bodies, and landscape heterogeneity, respectively. The information on the eco-epidemiological drivers for EGA can be central for disease control and prevention. Our method of linking land cover to disease risk may be applied to other vector-borne diseases and to other study regions.

Molecular evidence of Anaplasma phagocytophilum and Theileria equi coinfection in horses from Rio de Janeiro, Brazil

The present study aims to determine the frequencies of Theileria equi and Anaplasma phagocytophilum antibodies among horses from the state of Rio de Janeiro, Brazil, and to detect the presence of DNA of these pathogens through molecular methods. A total of 98 serum samples of horses from the municipality of Seropedica were tested by indirect immunofluorescence antibody (IFA) to detect anti-A. phagocytophilum and anti-T. equi IgG antibodies. In addition, quantitative real-time PCR (qPCR) was used to detect these pathogens in the DNA extracted from the whole blood and buffy coat of horses. Bivariate analysis and odds ratio were performed to verify the possible association between positivity and characteristics related to the horses. As evaluated by IFA and qPCR, the frequency of animals that tested positive for T. equi was 89.8% (n = 88/98) and 91.8% (n = 90/98), whereas A. phagocytophilum was 17.4% (n = 17/98) and 1.0% (n = 1/98), respectively. Serological evidence of exposure to A. phagocytophilum and T. equi was observed in 16.3% (n = 16/98) of the horses; however, exposure was confirmed by qPCR in only 1.0% (n = 1/98). No statistical association was found in the bivariate and odds ratio analysis. This is the first study reporting the molecular detection of A. phagocytophilum DNA in horses from the state of Rio de Janeiro, and also the coinfection of A. phagocytophilum and T. equi in a horse from Brazil confirmed by molecular methods. Equine granulocytic anaplasmosis is circulating in Brazilian horses, together with T. equi, and should be included in the differential diagnosis of tick-borne diseases.

Anaplasma phagocytophilum direct detection and exposure evidence in equines from two breeding farms from Minas Gerais State, Brazil

ABSTRACT: Equine granulocytic anaplasmosis is caused by Anaplasma phagocytophilum, a gram negative, obligatory intracellular bacterium, member of Anaplasmataceae family, included in the Rickettsiales order. Little is known about the disease, transmission dynamics, genetic diversity and prevalence in Minas Gerais state, Brazil. This work aimed to do a serosurvey using indirect immunofluorescent assay (IFA) test and evaluation of buffy coat smears, and nested polymerase chain reaction (PCR) as diagnostic methods, to determine the disease situation in horses from two manga-larga marchador breeding farms located in the municipalities of Ataléia e São Vicente de Minas, in Minas Gerais state. It was found that 76% (131/172) of the animals were considered reactive for IFA test, and the total of 12.8% was positive at buffy coat smears analysis. At PCR analysis, it was found 1.94% of the samples positive to the infection. Those samples were sequenced and showed 96% of similarity to A. phagocytophilum from a Ixodes ricinus tick. There is a high frequency of animals with the evidence of contact to A. phagocytophilum on the two evaluated properties in this study, which was proved by positiveness in PCR analysis. New researches must be carried out to better understand the epidemiologic and clinical dynamic of the disease in the state of Minas Gerais.

First molecular evidence of equine granulocytic anaplasmosis in Pakistan

Anaplasma phagocytophilum (A. phagocytophilum) is an obligate intracellular bacterium that causes equine granulocytic anaplasmosis (EGA) disease in equines. This pathogen has zoonotic potential, which makes it very important to be detected and controlled as early as possible. This study was aimed to assess the molecular prevalence, associated risk factors of EGA along with its effects on various hematological parameters. This study revealed an overall 10.67% prevalence in equine. Horses showed highest prevalence followed by mules and donkeys presenting 11.86, 10.53 and 9.43% prevalence, respectively. The samples were confirmed for anaplasmosis through sequencing. The BLAST queries confirmed very high homology of our isolates with Chinese and Japanese isolates of A. phagocytophilum (Accession no’s; KX505303, KY242456 and LC002836). The phylogenetic analysis found the study isolates clustered with each other and this cluster closely resembled Chinese isolate of A. bovis (FJ169957), A. phagocytophilum (HQ872464) and A. phagocytophilum (NR_044762) human isolate from northern Minnesota and Wisconsin. The key risk factors identified for occurrence of EGA in equine species on the basis of univariable analysis were sex of animal, housing type, tick infestation, previous tick history and tick control status, type of acaricides used, rearing system and farm hygiene, respectively. The hematological parameters like Hemoglobin (Hb), Total Leukocyte Count (TLC), Total Erythrocytes Count (TEC), and granulocytes were decreased in diseased animals. The mules showed no typical hematological variations which make sense for its nature as carrier of infection to the susceptible species. This is the first molecular evidence of EGA in Pakistan. The disease needs to be handled seriously as it has zoonotic potential. The animals should be properly attended in disease conditions as leukopenia, neutropenia and lymphopenia can aggravate the condition by making the animal prone to secondary infections.

SURVEY OF ANAPLASMA PHAGOCYTOPHILUM ANTIBODIES IN CAPTIVE PRZEWALSKI'S HORSES (EQUUS FERUS PRZEWALSKII).

Anaplasma phagocytophilum (formerly Ehrlichia equi ) is a tickborne pathogen of domestic horses and the causative agent of equine granulocytic anaplasmosis. After the occurrence of clinical anaplasmosis in a Przewalski's horse ( Equus ferus przewalskii) housed at the Smithsonian Conservation Biology Institute in 2008, opportunistic serosurveillance of the herd was initiated. From 2008 to 2014, 57 serum samples were collected from 27 individuals (10 males; 17 females). Using indirect immunofluorescent antibody assays for anti- Anaplasma phagocytophilum antibodies, it was determined that prevalence was 53%. No significant sex differences were identified. A statistical association between increasing age and seropositive status suggests cumulative risk of exposure to Anaplasma phagocytophilum . After exclusion of four clinical cases of anaplasmosis, it was found that 22-57% of those sampled each year were seropositive and clinically normal, suggesting that the majority of Przewalski's horses develop subclinical or self-limiting anaplasmosis after exposure to A. phagocytophilum .

Analysis of the Population Structure of Anaplasma phagocytophilum Using Multilocus Sequence Typing

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophils. It is transmitted via tick-bite and causes febrile disease in humans and animals. Human granulocytic anaplasmosis is regarded as an emerging infectious disease in North America, Europe and Asia. However, although increasingly detected, it is still rare in Europe. Clinically apparent A. phagocytophilum infections in animals are mainly found in horses, dogs, cats, sheep and cattle. Evidence from cross-infection experiments that A. phagocytophilum isolates of distinct host origin are not uniformly infectious for heterologous hosts has led to several approaches of molecular strain characterization. Unfortunately, the results of these studies are not always easily comparable, because different gene regions and fragment lengths were investigated. Multilocus sequence typing is a widely accepted method for molecular characterization of bacteria. We here provide for the first time a universal typing method that is easily transferable between different laboratories. We validated our approach on an unprecedented large data set of almost 400 A. phagocytophilum strains from humans and animals mostly from Europe. The typability was 74% (284/383). One major clonal complex containing 177 strains was detected. However, 54% (49/90) of the sequence types were not part of a clonal complex indicating that the population structure of A. phagocytophilum is probably semiclonal. All strains from humans, dogs and horses from Europe belonged to the same clonal complex. As canine and equine granulocytic anaplasmosis occurs frequently in Europe, human granulocytic anaplasmosis is likely to be underdiagnosed in Europe. Further, wild boars and hedgehogs may serve as reservoir hosts of the disease in humans and domestic animals in Europe, because their strains belonged to the same clonal complex. In contrast, as they were only distantly related, roe deer, voles and shrews are unlikely to harbor A. phagocytophilum strains infectious for humans, domestic or farm animals.

Comparative analysis of 16S RNA nucleotide sequences of Anaplasma phagocytophilum detected in the blood of horses from various parts of Europe

The aim of this study was to conduct a comparative analysis of the 16S rRNA gene fragment nucleotide sequences for Anaplasma phagocytophilum strains detected in the blood of horses from various parts of Europe. The study comprised 234 horses that had had contact with ticks. Using PCR, the genetic material of A. phagocytophilum was identified in the blood of 42 animals. The sequences of the 16S RNA gene amplicons that were obtained from our A. phagocytophilum isolates had 100 % similarity with each other and 96.4-100 % similarity with Anaplasma spp. sequences selected from those available in GenBank. Nucleotide substitutions at positions 248 and 249 were demonstrated in all the 16S RNA gene sequences of Anaplasma obtained in our study. This may indicate the emergence of a new rickettsial genotype that is the cause of equine granulocytic anaplasmosis in southern and eastern Europe. These results add new information on the epidemiology and genetic diversity of A. phagocytophilum detected in the blood of horses from southern and eastern Europe.