Anaplasma marginale infection in a Japanese Black cow 13 years after eradication of Rhipicephalus (Boophilus) microplus in Okinawa, Japan

In October 2007, a 15-year-old Japanese Black cow on Ishigaki Island, Okinawa, Japan, was diagnosed with Anaplasma marginale infection based on clinical symptoms, blood examination, smear observation, 16S rRNA and groEL gene sequence analysis, and the result of a CF test. The cow was introduced into the farm from mainland Japan as a calf in 1993, one year before the eradication of Rhipicephalus (Boophilus) microplus, the main vector of A. marginale in Okinawa Prefecture. It is possible that the cow was first infected with A. marginale as a calf in Ishigaki Island and had been persistently infected since then. This is the first reported clinical case of A. marginale infection of cattle since the eradication of R. microplus in Okinawa Prefecture. Additional analysis of major surface protein 1α amino acid sequences revealed that the A. marginale Okinawa strain presented four new repeat forms which were not seen in other strains. This indicates that the Okinawa strain may be a unique geographical variant of A. marginale.     

Outbreak of anaplasmosis associated with novel genetic variants of Anaplasma marginale in a dairy cattle

During early lactation, dairy cows may present a transient immunosuppressive state and develop anaplasmosis caused by Anaplasma marginale. In this study, clinical anaplasmosis in dairy cattle in the Thrace region of Turkey was investigated with respect to within-herd prevalence, vertical transmission, and genetic diversity. In March and September 2015, thirty lactating cows showed primary clinical signs of anaplasmosis, including fever, anaemia, decreased milk yield, anorexia, and laboured breathing. Symptoms disappeared in most cows after administration of long-acting oxytetracycline, but nine of them (30%) died. Following diagnosis based on clinical signs, microscopy and molecular findings, blood samples were collected from apparently healthy lactating cows (n = 184), pregnant heifers (n = 39) and newborn calves (n = 24). DNA was extracted from each sample and analyzed for the presence of major surface proteins (MSPs) of A. marginale, followed by sequencing to assess diversity of isolates. Microscopic examination of erythrocytes revealed A. marginale inclusion bodies in symptomatic cows. Examination of thin blood smears showed 3.8% of the lactating, clinically asymptomatic, cows to be infected with A. marginale, while nPCR detected 31.0% positive. A. marginale infection was not detected in pregnant heifers by either method. Congenital infection was found in one calf by nPCR. This is the first report of transplacental transmission of A. marginale in Turkey. The MSP4 sequence analyses showed high genetic diversity among the isolates, presenting 97.6-99.6% homology at the amino acid level. The sequences of MSP1a amplicons revealed genetic diversity providing three new tandem repeats.     

A technique to establish a laboratory colony of Boophilus microplus infected with Babesia bigemina

A technique was evolved for the establishment and maintenance of a colony Boophilus microplus free of infection with Anaplasma marginale and Babesia spp., and for their subsequent infection with a pure isolate of Babesia bigemina. Confirmation was obtained that the ticks are infected normally during the last 24 h of attachment on the host. The life cycle of Boophilus microplus was described for a single situation on the Atlantic Coast of Colombia.     

The natural history of Anaplasma marginale

The intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), described by Sir Arnold Theiler in 1910, is endemic worldwide in tropical and subtropical areas. Infection of cattle with A. marginale causes bovine anaplasmosis, a mild to severe hemolytic disease that results in considerable economic loss to both dairy and beef industries. Transmission of A. marginale to cattle occurs biologically by ticks and mechanically by biting flies and by blood-contaminated fomites. Both male ticks and cattle hosts become persistently infected with A. marginale and serve as reservoirs of infection. While erythrocytes are the major site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks that begins by infection of gut cells, and transmission to susceptible hosts occurs from salivary glands during feeding. Major surface proteins (MSPs) play a crucial role in the interaction of A. marginale with host cells, and include adhesion proteins and MSPs from multigene families that undergo antigenic change and selection in cattle, thus contributing to maintenance of persistent infections. Many geographic strains of A. marginale have been identified worldwide, which vary in genotype, antigenic composition, morphology and infectivity for ticks. Isolates of A. marginale may be maintained by independent transmission events and a mechanism of infection/exclusion in cattle and ticks. The increasing numbers of A. marginale genotypes identified in some geographic regions most likely resulted from intensive cattle movement. However, concurrent A. marginale strain infections in cattle was reported, but these strains were more distantly related. Phylogenetic studies of selected geographic isolates of A. marginale, using msp4 and msp1α, provided information about the biogeography and evolution of A. marginale, and msp1α genotypes appear to have evolved under positive selection pressure. Live and killed vaccines have been used for control of anaplasmosis and both types of vaccines have advantages and disadvantages. Vaccines have effectively prevented clinical anaplasmosis in cattle but have failed to block A. marginale infection. Vaccines are needed that can prevent clinical disease and, simultaneously, prevent infection in cattle and ticks, thus eliminating these hosts as reservoirs of infection. Advances in genomics, proteomics, immunology and biochemical and molecular technologies during the last decade have been applied to research on A. marginale and related organisms, and the recent development of a cell culture system for A. marginale has provided a format for studying the pathogen/tick interface. Recent advancements and new research methodologies should provide additional opportunities for development of new strategies for control and prevention of bovine anaplasmosis.     

Targeting the Tick/Pathogen Interface for Developing New Anaplasmosis Vaccine Strategies

Bovine anaplasmosis is a tick-borne hemolytic disease of cattle that occurs worldwide caused by the intraerythrocytic rickettsiae Anaplasma marginale. Control measures, including use of acaricides, administration of antibiotics and vaccines, have varied with geographic location. Our research is focused on the tick-pathogen interface for development of new vaccine strategies with the goal of reducing anaplasmosis, tick infestations and the vectorial capacity of ticks. Toward this approach, we have targeted (1) development of an A. marginale cell culture system to provide a non-bovine antigen source, (2) characterization of an A. marginale adhesion protein, and (3) identification of key tick protective antigens for reduction of tick infestations. A cell culture system for propagation of A. marginale was developed and provided a non-bovine source of A. marginale vaccine antigen. The A. marginale adhesion protein, MSP1a, was characterized and use of recombinant MSP1a in vaccine formulations reduced clinical anaplasmosis and infection levels in ticks that acquired infection on immunized cattle. Most recently, we identified a tick-protective antigen, subolesin, that reduced tick infestations, as well as the vectorial capacity of ticks for acquisition and transmission of A marginale. This integrated approach to vaccine development shows promise for developing new strategies for control of bovine anaplasmosis.     

Experimental immunization of calves against Anaplasma marginale infection: observations on the use of living A. centrale and A. marginale

Groups of Bos indicus cross calves aged 6 months were immunized with Anaplasma centrale or A. marginale. All animals were challenged with 10¹⁰A. marginale 7 months later. Groups immunized with A. marginale were refractory to challenge, whereas only a portion of the animals vaccinated with A. centrale were immune. Following immunization, animals that had experienced a good primary antibody response as measured by a complement fixation test, a marked reduction in packed cell volume, and a high parasitaemia, resisted a subsequent challenge with A. marginale. Resistance was characterised by a weak secondary antibody response and the absence of A. marginale in blood films     

A case of apparent suppression of Anaplasma marginale infection by eperythrozoonosis (Eperythrozoon teganodes)

The apparent suppression of Anaplasma marginale infection by Eperythrozoon teganodes in a splenectomized calf has been reported. A splenectomized calf, inoculated with 500 ml of blood having 23% erythrocytes infected with A. marginale, developed eperythrozoonosis on the fourth day post inoculation. A. marginale parasitaemia remained very low during the patent eperythrozoonosis. A. marginale parasites started to increase in number only after E. teganodes infection had been controlled with neoarsphenamine. A splenectomized calf treated identically, but not showing E. teganodes parasites in the peripheral blood, developed clinical anaplasmosis and fulminant parasitaemia within 3-4 days post inoculation.     

Comparison of rapid card agglutination test with the complement fixation test for diagnosis of Anaplasma marginale infection in Cattle in Colombia

Three hundred and thirty-nine serum samples collected from nine Holstein-Friesian calves before and during natural infection with Anaplasma marginale on the north coast of Colombia were used to compare the complement fixation (CF) and rapid card agglutination tests (CT) for the diagnosis of anaplasmosis. The positive agglutination of the CT always developed several days after the first CF reaction but then persisted. In contrast, the CF test showed an initial sharp rise in titer but then showed fluctuations between trace and 1 : 80 titers. The results indicated that under field conditions the CT was the simpler and more reliable test for the diagnosis of anaplasmosis in a single animal. The CF test remains useful in experimental situations where the earliest knowledge is required of the magnitude of immunological response to challenge.     

Validation of a multiplex PCR assay to detect Babesia spp. and Anaplasma marginale in cattle in Uruguay in the absence of a gold standard test

Detection of bovine Babesia spp. and Anaplasma marginale is based on the reading of Giemsa-stained blood or organ smears, which can have low sensitivity. Our aim was to improve the detection of bovine Babesia spp. and A. marginale by validating a multiplex PCR (mPCR). We used 466 samples of blood and/or organs of animals with signs and presumptive autopsy findings of babesiosis or anaplasmosis. The primers in our mPCR amplified the rap-1a gene region of Babesia bovis and B. bigemina, and the msp-5 region of A. marginale. We used a Bayesian model with a non-informative priori distribution for the prevalence estimate and informative priori distribution for estimation of sensitivity and specificity. The sensitivity and specificity for smear detection of Babesia spp. were 68.6% and 99.1%, and for A. marginale 85.6% and 98.8%, respectively. Sensitivity and specificity for mPCR detection for Babesia spp. were 94.2% and 97.1%, and for A. marginale 95.2% and 92.7%, respectively. Our mPCR had good accuracy in detecting Babesia spp. and A. marginale, and would be a reliable test for veterinarians to choose the correct treatment for each agent.     

Anaplasmosis in Cattle in Italy

Bovine anaplasmosis caused by Anaplasma marginale is a disease transmitted by ticks belonging to the Ixodidae family. Southern Italy is considered an endemic zone but environmental and social factors are changing the epidemiology of the disease to expand to previously anaplasmosis-free regions. The available data of published reports of anaplasmosis in Italy together with the data obtained by the National Centre of Reference for Anaplasma, Babesia, Rickettsia and Theileria (C.R.A.Ba.R.T.), allowed to report A. marginale infection in different Italian regions (Lazio, Marche, Campania, Puglia, Basilicata, Calabria, Lombardy, Tuscany, Umbria and Sicily). Cattle are also subject to infection with the related Ixodes ricinus-transmitted pathogen, Anaplasma phagocytophilum that results in reduced milk production in cattle. A. phagocytophilum infect also small ruminants, domestic and wild animals and causes the human granulocytic anaplasmosis. Different studies have been conducted on the presence of A. phagocytophilum in Italy both in the tick vectors and in the wild and domestic reservoirs. Contrary to A. marginale, the prevalence of A. phagocytophilum embraces the whole Italian territory from the Alps to the southern and insular regions.     

A serological survey for anaplasmosis in cattle in the Mexican states of Nuevo Leon,Tamaulipas and Coahuila using the card test

Blood samples from 2156 cattle from 40 herds in the Mexican states of Nuevo Leon, Tamaulipas and Coahuila were tested for antibody activity to Anaplasma marginale using the card test. Herd prevalence rates ranged from 0 to 86% with a mean of 22%. Analysis of joint positivity to A. marginale and Babesia bovis and to A. marginale and B. bigemina in individual animals resulted in the rejection of the null hypothesis of no association in 7 and 8 of 37 herds, respectively.     

Characterization of Anaplasma phagocytophilum and A. ovis infection in a naturally infected sheep flock with poor health condition

Anaplasma species are transmitted by ticks and cause diseases in humans and animals. These pathogens infect sheep, an economically important domestic animal worldwide. The current study was designed to characterize in 200 animals the infection with Anaplasma phagocytophilum and Anaplasma ovis and the genetic diversity of A. ovis strains collected from a naturally infected sheep flock with poor health condition. Sheep had 98% seroprevalence to Anaplasma spp. antibodies. PCR results confirmed the presence of A. phagocytophilum and A. ovis DNA in 11.5% and 37% of the sheep, respectively. Concurrent infections were detected in 6.5% of the sheep. Seventy-one adult ticks were collected from 45 sheep with infestations ranging from one to 15 ticks per animal. The analysis of A. ovis msp4 sequences demonstrated a previously unreported polymorphism for this pathogen with 17 different haplotypes in infected sheep. These results demonstrated that, although A. ovis msp4 haplotypes may be less variable when compared with Anaplasma marginale and A. phagocytophilum strains on a global scale, genetic polymorphisms occur in this locus in strains obtained from an infected sheep flock with poor health condition.     

Serological responses to bovine anaplasmosis following treatment with gloxazone

Groups of unsplenectomised steers and calves were infected with either the Onderstepoort or Sukari 1 strain of Anaplasma marginale by the injection of infected blood. The infections were treated with gloxazone (2.5 – 25 mg/kg). The serological response of the animals was monitored by the complement fixation (CF) and capillary tube agglutination (CA) tests, using antigens prepared from the Onderstepoort strain. CF antibody responses were alike in both strains. The titre rose rapidly from four days after infection to a peak around the time of the initial crisis. These high titres were maintained, and generally did not rise further at the time of recrudescence of parasitaemia. CA antibody appeared 7–10 days after infection and reached a peak shortly after the initial crisis. Titres fell as parasitaemia was reduced following gloxazone treatment, then rose as parasitaemia recrudesced. There was a close correlation between the effectiveness of gloxazone treatment and changes in CA antibody titre.     

A new PCR-RFLP method for detection of <Emphasis Type="Italic">Anaplasma marginale</Emphasis> based on 16S rRNA

Species of the genus Anaplasma (Rickettsiales: Anaplasmataceae) are obligate intracellular tick borne pathogens. Three species of Anaplasma that infect cattle and sheep (A. marginale, A. centrale and A. ovis) are well recognized. Of these erythrocytic Anaplasma, A. marginale can cause diseases in the livestock with high economical losses. Species-specific PCR based on 16S rRNA gene is commonly used for detection of Anaplasma species but can not differentiate A. marginale, A. centrale and A. ovis because of sequence similarity. In this study DNA extraction was performed on 50 blood samples with presence of Anaplasma spp. in marginal point of erythrocytes in their blood smears. The extracted DNA from blood cells was analyzed by PCR and PCR-RFLP using primers derived from 16S rRNA gene and restriction endonuclease Bst1107 I. The restriction endonuclease Bst1107I only recognizes the sequence (GTATAC) in corresponding PCR product of A. marginale and cut it. The nucleotide sequence of the A. marginale 16S rRNA gene was determined and compared with the sequences of A. marginale in GenBank. The 16S rRNA of A. marginale in Iran was completely similar to the related sequence deposited in GenBank at accession number of M60313. In the present study we propose a new PCR-restriction fragment length polymorphism analysis (RFLP) method based on 16S rRNA gene for specific detection of A. marginale.     

Assessment of a low virulence Australian isolate of Anaplasma marginale for pathogenicity, immunogenicity and transmissibility by Boophilus microplus

A 14-year-old cow (Dawn) born and kept in a Boophilus microplus-free region gave birth to a calf, which showed the presence of an Anaplasma marginale infection after splenectomy. The calf's grand dam was from a B. microplus infected area and we assume the infection originated via the transplacental route over two generations. An isolate, prepared from the calf, had similar or lower pathogenicity as Anaplasma centrale, and previously exposed steers were resistant to challenge by four A. marginale field isolates. Two attempts to transmit the isolate using B. microplus were unsuccessful. Our results indicate that Dawn A. marginale may be a useful vaccine in Australia and warrants larger scale validation of its safety and potency locally as well as of the protection it affords against African and New World isolates.     

The use of tick transmission by Boophilus microplus to isolate pure strains of Babesia bovis,Babesia bigemina and Anaplasma marginale from cattle with mixed infections

Pure strains of Babesia bovi, Babesia bigemina and Anaplasma marginale were isolated from cattle infected with all 3 species as well as a Theileria sp. and Eperythrozoon teganodes, using only transmission by the tick, Boophilus microplus. Unengorged adult ticks transferred to susceptible cattle transmitted A. marginale, but not Babesia. Engorged adults gave rise to progeny that transmitted Babesia, B. bovis by larvae and B. bigemina by male ticks. The Theileria and E. teganodes were not transmitted by the ticks and thus did not appear in calves used for isolating the pure strains of Babesia and A. marginale.     

Risk factors for anaplasmosis in dairy cows during the peripartum

Anaplasma marginale is endemic in tropical and subtropical areas around the world. Some studies have suggested that cows during peripartum may present a transient immunosuppression state and development of clinical signs of anaplasmosis. The aim of this study was to investigate the relationship between some risk factors and the seroprevalence of A. marginale in dairy cows during peripartum in Rio de Janeiro, Brazil. The risk factors analyzed in association with the prevalence of antibodies against A. marginale in dairy cows were calving season, reproductive experience, breed standard, tick infestations, stocking density, and milk yield. The antibodies against A. marginale were tested in indirect enzyme-linked immunosorbent assays. A primary screening using a 2 × k contingency table of the exposed variables with the outcomes was performed. All variables for which p?<?0.20 were included in a fixed effects log regression. The risk factors investigated to anaplasmosis were calving (OR 2.61, IC 1.08–7.63), breed standard (OR 3.83, IC 0.08–0.28), reproductive experience (OR 33.7, IC 2.14–5.16), milk yield (OR 3.9, IC 2.24–7.03), Rhipicephalus microplus infestations (OR 10.3, IC 0.05–0.17), and stocking density (OR 22.3, IC 0.05–0.17). Low titers of antibodies against A. marginale during peripartum had been characterized as a period previous to development of clinical anaplasmosis. Thus, studies on anaplasmosis should consider each farm as an epidemiological unit, where environmental and immunological factors may influence the endemic status of the pathogen.     

Transformation of Anaplasma marginale

The tick-borne pathogen, Anaplasma marginale, has a complex life cycle involving ruminants and ixodid ticks. It causes bovine anaplasmosis, a disease with significant economic impact on cattle farming worldwide. The obligate intracellular growth requirement of the bacteria poses a challenging obstacle to their genetic manipulation, a problem shared with other prokaryotes in the genera Anaplasma, Ehrlichia, and Rickettsia. Following our successful transformation of the human anaplasmosis agent, A. phagocytophilum, we produced plasmid constructs (a transposon bearing plasmid, pHimarAm-trTurboGFP-SS, and a transposase expression plasmid, pET28Am-trA7) designed to mediate random insertion of the TurboGFP and spectinomycin/streptomycin resistance genes by the Himar1 allele A7 into the A. marginale chromosome. In these trans constructs, expression of the fluorescent and the selectable markers on the transposon, and expression of the transposase are under control of the A. marginale tr promoter. Constructs were co-electroporated into A. marginale St. Maries purified from tick cell culture, and bacteria incubated for 2 months under selection with a combination of spectinomycin and streptomycin. At that time, ≤1% of tick cells contained colonies of brightly fluorescent Anaplasma, which eventually increased to infect about 80–90% of the cells. Cloning of the insertion site in E. coli and DNA sequence analyses demonstrated insertion of the entire plasmid pHimarAm-trTurboGFP-SS encoding the transposon in frame into the native tr region of A. marginale in an apparent single homologous crossover event not mediated by the transposase. Transformants are fastidious and require longer subculture intervals than wild type A. marginale. This result suggests that A. marginale, as well as possibly other species of Anaplasma and Ehrlichia, can be transformed using a strategy of homologous recombination.     

Cloning, expression, and characterization of the MSP1a and MSP1b recombinant proteins from PR1 Anaplasma marginale strain, Brazil

The Anaplasma marginale is a bacterium that has obligate intraerythrocytic multiplication in cattle causing important economic loss. The A. marginale major surface protein 1 (MSP1) complex, heterodimer composed of MSP1a and MSP1b, has been identified as adhesins for bovine erythrocytes. The objectives of this study were to sequences the msp1β gene and produce and characterize recombinant MSP1a and MSP1b from a Brazilian strain of A. marginale, PR1. The msp1α and msp1β genes from the PR1 strain were cloned and expressed in E. coli BL21 Star using the vectors pET102 and pET101/D-TOPO. Antibodies were produced against the recombinant proteins and were shown to react with rMSP1a and rMSP1b demonstrating a molecular mass of 70 kDa to 105 kDa and 100 kDa, respectively for these proteins. Bovine erythrocytes were agglutinated by BL21/rMSP1a and BL21/rMSP1b and, this agglutination was inhibited by the presence of the IgY anti-rMSP1a, confirming the adhesion function of these proteins. Additionally, using the IgY anti-rMSP1a and rMSP1b in a IFI, the presence of rMSP1a and rMSP1b was confirmed on the outer membrane of the recombinant E. coli BL21. Our results show that the msp1β gene from the PR1 strain has both the conserved region and contain the defined polymorphism regions previously described for other strains of A. marginale. The results from this study confirm adhesive functions for rMSP1a and rMSP1b from PR1 strain in bovine erythrocytes invasion.     

Vaccination withanaplasma centrale: Response after an experimental challenge withanaplasma marginale

Summary The haematological and clinical responses to vaccination withAnaplasma centrale and to subsequent challenge withAnaplasma marginale were evaluated. Twenty Holstein steers 14 to 16 months of age were divided into two groups of 12 and eight animals respectively (groups I and II). Group I was inoculated on day zero with 10⁷ A. centrale-infected erythrocytes and group II was kept as a control. On day 125 both groups were challenged with 5 × 10⁷ A. marginale-infected erythrocytes.A. centrale inoculation produced low parasitaemias (maximum -x 2.7%), moderate packed cell volume (PCV) falls (minimum -x 20.5%) and no clinical symptoms. After the challenge group I had significantly lower parasitaemia (maximum -x 2.3%) and higher PCV (minimum -x 20.1%) than group II (7.5% and 14.5% respectively). Four steers from group II developed acute anaplasmosis and required treatment.

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Vacunacion ConAnaplasma Centrale: Respuesta Despues del Desafio Experimental ConAnaplasma Marginale

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Vaccination AvecAnaplasma Centrale: Resultats Apres une Confrontation Experimentale AvecA. Marginale

Résumé Les auteurs ont évalué les résultats hématologiques et clinique de la vaccination avecAnaplasma centrale et secondairement ceux d'un essai comparatif expérimental avecAnaplasma marginale. Vingt bouvillons Holstein agés de 14 à 16 mois ont été divisés en deux groupes (I et II) respectivement de 12 et 8 sujets. Le groupe I a re?u au jour J⁰ une injection de 10⁷ érytrhocytes infectés parAnaplasma centrale. Le groupe II a servi de lot témoin. Au 125 ème jour les deux groupes ont été soumis à une injection de 5 × 10⁷ érythrocytes infectés parAnaplasma marginale. L'injection d'A. centrale a produit une parasitémie légère (maximum -x 2,7 p.100), une chute modérèe du volume de l'Hématocrite (PVC) (minimum -x 20,5 p.100) et aucun sympt?me clinique. Par rapport au groupe II et après l'épreuve expérimentale, le groupe I présentait significativement une parasitémie plus basse (maximum -x 2,3 p.100) et de taux plus élevés (minimum -x 20,1 p.100), contre 7,5 et 14,5 p.100 respectivement. Quatre bouvillons du groupe II ont été atteints d'une anaplasmose aigüe exigeant un traitement. Resumen Se evaluó la respuesta clínica y hematológica despues de la vacunación conA. centrale, seguida del desafío conA. marginale. Un grupo de novillos Holstein de 14 a 16 meses de edad, se dividieron en dos grupos de 12 y ocho animales respectivamente (grupos I y II). El grupo I se inoculó el día cero con 10⁷ eritrocitos infectados conA. centrale, sirviendo el grupo II de testigo. El día 125, ambos grupos recibieron una descarga de 5 × 10⁷ eritrocitos infectados conA. marginale. La inoculación conA. centrale produjo parasitémias bajas (máximo -x 2.7%), caídas moderadas del volumen corpuscular (PCV), (mínimo -x 20.5%) y no se presentaron síntomas clínicos, Despues de la descarga, el grupo I tuvo parasitémias significativamente más bajas (máximo -x 2.3%) y volumenes corpusculares (PCV) más altos (mínimo 20.1%) que el grupo II (7.5% y 14.5% respectivamente). Cuatro novillos del grupo II desarrollaron anaplasmosis aguda y fueron tratados.