Genetic diversity of Anaplasma marginale strains from an outbreak of bovine anaplasmosis in an endemic area

Anaplasma marginale is a tick-borne pathogen of cattle that causes the disease bovine anaplasmosis worldwide. Major surface proteins (MSPs) are involved in host-pathogen and tick-pathogen interactions and have been used as markers for the genetic characterization of A. marginale strains. A. marginale genotypes are highly variable in endemic areas worldwide. The genetic composition of A. marginale strains during anaplasmosis outbreaks has been characterized in one study only which reported a single msp1alpha genotype in infected cattle. However, more information is required to characterize whether a single genotype is responsible for an anaplasmosis outbreak or whether multiple genotypes can cause disease in na?ve cattle within a single herd in endemic areas. The aim of this study was to characterize the genetic diversity of A. marginale strains from an outbreak of bovine anaplasmosis in the State of Tamaulipas, Mexico. A. marginale genotypes were characterized at the molecular level using msp4 and msp1alpha gene sequences. The results revealed that several A. marginale genotypes are present in cattle during acute anaplasmosis outbreaks, thus suggesting that mechanical transmission or stochastic biological transmission through equally efficient independent transmission events may explain A. marginale genotype frequency in a cattle herd during acute bovine anaplasmosis outbreaks in endemic areas. The results reported herein corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. The development and implementation of anaplasmosis control measures is dependent upon understanding the epidemiology of A. marginale in endemic regions, including the characterization of the genetic diversity of strains that produce outbreaks of bovine anaplasmosis.     

Prevalence and genetic diversity of Anaplasma marginale strains in cattle in South Africa

Bovine anaplasmosis, caused by the tick-borne rickettsia Anaplasma marginale, is endemic in South Africa and results in considerable economic loss to the cattle industry. This study was designed to characterize strains of A. marginale at the molecular level from cattle raised in communal and commercial farms in the north-eastern and south-western regions of the Free State Province, South Africa, that varied in rainfall and vegetation. Seroprevalence to A. marginale was determined in 755 cattle by an Anaplasma spp. competitive enzyme-linked immunosorbent assay and ranged from 44% to 98% and was similar in both regions. While Anaplasma centrale was not targeted in this study, A. marginale infections were identified by species-specific msp1alpha polymerase chain reaction in 129 of 215 of the samples studied. Similar genetic diversity of A. marginale strains was found in both the north-eastern and south-western regions. The sequences of 29 A. marginalemsp1alpha amplicons from South African strains revealed considerable genetic diversity providing 14 new repeat sequences. However, 42% of MSP1a repeat sequences were not unique to this region. These results indicated the presence of common genotypes between South African, American and European strains of A. marginale. Cattle movement between different parts of South Africa was suggested by the presence of identical A. marginale MSP1a genotypes in north-eastern and south-western regions of the Free State Province. Control strategies for anaplasmosis in South Africa should therefore be designed to be protective against genetically heterogeneous strains of A. marginale.     

Phylogenetic analysis of Anaplasma marginale strains from Paraná State, Brazil, using the msp1alpha and msp4 genes

Anaplasma marginale is an obligate intraerythrocytic rickettsial pathogen (order, Rickettsiales: family, Anaplasmataceae) that causes bovine anaplasmosis. This disease is widely distributed in tropical and sub-tropical regions of the world and causes important economic losses to cattle production. Major surface protein (MSP)1a (msp1alpha gene) is one of the six MSPs identified on A. marginale from cattle, whose sequence and size vary according to the number of tandem 28- to 29-amino acid repeats. This study characterized the msp1alpha and msp4 genes obtained from three distinct Brazilian herds from the State of Paraná. Three strains of the msp1alpha and one strain of the msp4 gene were sequenced. The strains evaluated revealed PCR products of different size, representing three, five and six internal repeats. Sequence analyses confirmed the number of tandem sequence copies and revealed a high degree of sequence identity with strains from other Brazilian States, as well as strains from the USA, Europe and Israel. The msp1alpha DNA and amino acid sequences from A. marginale and DNA sequences of msp4 strains did not reveal distinct phylogeographical segregation. However, the amino acid sequences of msp4 demonstrated definite phylogeographical relationship. These results suggest that the amino acid sequences of msp4 should be used for phylogenetic identification of A. marginale strains and may be an important tool for the epidemiology and control of anaplasmosis. Additionally, the close similarity of the Paraná strains of A. marginale with strains from USA, Europe and Asia may reflect the introduction of these genes during the development of the Brazilian bovine herd.     

Serologic and molecular characterization of Anaplasma species infection in farm animals and ticks from Sicily

Although Anaplasma marginale was known to be endemic in Italy, the diversity of Anaplasma spp. from this area have not been characterized. In this study, the prevalence of Anaplasma spp. antibodies in randomly selected farm animals collected on the island of Sicily was determined by use of a MSP5 cELISA for Anaplasma spp. and an immunofluorescence test specific for Anaplasma phagocytophilum. Genetic variation among strains of Anaplasma spp. from animals and ticks was characterized using the A. marginale msp1alpha and the Anaplasma spp. msp4 genes. Eight species of ticks were collected and tested by PCR. Seropositivity for Anaplasma spp. and A. phagocytophilum was detected in bovine and ovine samples. All the donkeys were seropositive for A. phagocytophilum but not for Anaplasma spp. Four A. marginale genotypes were identified by msp4 sequences from bovine and tick samples. Two new genotypes of Anaplasma ovis were characterized in sheep. The sequences of A. phagocytophilum from three donkeys proved to be identical to the sequence of the MRK equine isolate from California. Six A. marginale genotypes were found in cattle and one tick using the A. marginale msp1alpha sequences. All genotypes had four repeated sequences in the N-terminal portion of the MSP1a, except for one that had five repeats. The Italian strains of A. marginale contained three repeat sequences that were not reported previously. Definition of the diversity of Anaplasma spp. in Sicily reported, herein is fundamental to development of control strategies for A. marginale, A. ovis and A. phagocytophilum in Sicily.     

Concomitant infection of cattle with the vaccine strain Anaplasma marginale ss centrale and field strains of A. marginale

Bovine anaplasmosis, caused by Anaplasma marginale, the intraerythrocytic rickettsia, is controlled by vaccination with live Anaplasma marginale ss centrale (A. centrale), a subspecies of relatively low pathogenicity. We have experimentally demonstrated that an animal primarily infected with A. marginale, or with the related vaccine subspecies A. centrale can be infected with the heterologous subspecies, and carries both bacteria. The co-infection was detected in experimentally cross-infected calves for up to 3 months after the last inoculation with the heterologous subspecies. The occurrence of characteristic cyclic rickettsemia of A. centrale and A. marginale was observed by examination of Giemsa-stained blood smears, or by the presence of specific rickettsial DNA confirmed in PCR assays based on specific msp1a and msp4 for A. marginale, and on specifically designed msp3 and msp4 primers for A. centrale. Sequence analysis of msp4-specific fragments for each subspecies revealed the presence of dual infection in both calves on days 30 and 60 after cross-inoculation with the heterologous Anaplasma subspecies. The experimental cross-infection of calves clearly demonstrated that the concept of "infection exclusion" does not apply to Anaplasma infection in cattle; as there was no infection exclusion of A. marginale in A. centrale-infected cattle, and vice versa. The present results confirmed our previous findings that cattle grazing in an anaplasmosis-endemic field were subject to concomitant infection with both the vaccine A. centrale and the field A. marginale strains.     

Molecular phylogeny and biogeography of North American isolates of Anaplasma marginale (Rickettsiaceae: Ehrlichieae)

Anaplasma marginale (A. marginale) is a tick-borne ehrlichial pathogen of cattle that causes the disease anaplasmosis. Six major surface proteins (MSPs) have been identified on A. marginale from cattle and ticks of which three, MSP1a, MSP4 and MSP5, are from single genes and do not vary within isolates. The other three, MSP1b, MSP2 and MSP3, are from multigene families and may vary antigenically in persistently infected cattle. Several geographic isolates have been identified in the United States which differ in morphology, protein sequence and antigenic properties. An identifying characteristic of A. marginale isolates is the molecular weight of MSP1a which varies in size among isolates due to different numbers of tandemly repeated 28-29 amino acid peptides. For these studies, genes coding for A. marginale MSP1a and MSP4, msp1alpha and msp4, respectively, from nine North American isolates were sequenced for phylogenetic analysis. The phylogenetic analysis strongly supports the existence of a south-eastern clade of A. marginale comprised of Virginia and Florida isolates. Analysis of 16S rDNA fragment sequences from the A. marginale tick vector, Dermacentor variabilis, from various areas of the United States was used to evaluate possible vector-parasite co-evolution. Our phylogenetic analysis supports identity between the most parsimonious tree from the A. marginale MSP gene data and the tree that reflected the western and eastern clades of D. variabilis. These phylogenetic analyses provide information that may be important to consider when developing control strategies for anaplasmosis in the United States.     

Experimental transmission of field Anaplasma marginale and the A. centrale vaccine strain by Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus ticks

The cattle rickettsia Anaplasma marginale is distributed worldwide and is transmitted by about 20 tick species, but only Rhipicephalus simus, a strictly African tick species, has been shown to transmit the vaccine strain of A. centrale. The aim of the present study was to examine transmission of field strains of A. marginale and of the vaccine strain of A. centrale by three tick species -Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus - to susceptible calves. Two genetically distinct Israeli field strains of A. marginale, tailed and non-tailed (AmIsT and AmIsNT, respectively), were efficiently transmitted by R. sanguineus, whereas H. excavatum transmitted only the tailed isolate, and R. (Boophilus) annulatus did not transmit A. marginale. None of the three tick species transmitted A. centrale. By means of msp1a primers in PCR assays, amplicons of similar sizes were obtained from either A. marginale-infected calves that were used for acquisition feeding, from R. sanguineus fed on the infected calves, or from calves to which anaplasmosis had been successfully transmitted by these ticks. Although an A. centrale-specific fragment was amplified from salivary glands of R. sanguineus, no transmission to susceptible cattle occurred during 3 months of observation, and anaplasmosis was not induced in splenectomized calves that were subinoculated with blood from calves on which R. sanguineus had fed.     

First serological and molecular evidence on the endemicity of Anaplasma ovis and A. marginale in Hungary

Recurring and spontaneously curing spring haemoglobinuria was recently reported in a small sheep flock in a selenium deficient area of northern Hungary. In blood smears of two animals showing clinical signs, Anaplasma-like inclusion bodies were seen in erythrocytes. To extend the scope of the study, 156 sheep from 5 flocks and 26 cattle from 9 farms in the region were examined serologically with a competitive ELISA to detect antibodies to Anaplasma marginale, A. centrale and A. ovis. The seropositivity in sheep was 99.4%, and in cattle 80.8%. A. ovis and A. marginale were identified by PCR and sequence analysis of the major surface protein (msp) 4 gene in sheep and cattle, respectively. Haemoglobinuria, an unusual clinical sign for anaplasmosis might have been a consequence of transient intravascular haemolysis facilitated by selenium deficiency in recently infected sheep, as indicated by the reduction of mean corpuscular haemoglobin concentration (MCHC). Membrane damage was also demonstrated for parenchymal cells, since their enzymes showed pronounced elevation in the plasma. Ticks collected from animals in the affected as well as in neighbouring flocks revealed the presence of Dermacentor marginatus, Ixodes ricinus and D. reticulatus, with the dominance of the first. The present data extend the northern latitude in the geographical occurrence of ovine anaplasmosis in Europe and reveal the endemicity of A. ovis and A. marginale in Hungary.     

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.     

Genetic diversity and molecular phylogeny of Anaplasma marginale isolates from Minas Gerais, Brazil

Anaplasma marginale (Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world, and many isolates of A. marginale may occur in a given geographic area. Phylogenetic relationships have been reported for A. marginale isolates from the US using gene and protein sequences of MSP1a and msp4. These studies demonstrated that msp4 sequences, but not MSP1a DNA or protein sequences, provide phylogeographic information and also that MSP1a sequences are highly heterogeneous among A. marginale populations. However, little information is available on the genetic diversity of A. marginale isolates from other regions of the world. The present study was undertaken to examine genetic variation among 10 isolates of A. marginale obtained from infected cattle in the State of Minas Gerais, Brazil, where A. marginale is endemic. Neighbor-joining analysis of msp4 sequences of Brazilian and New World isolates of A. marginale from Argentina, Mexico and the US provided bootstrap support for a Latin American clade. The sequences of the MSP1a repeats of four Brazilian isolates of A. marginale were compared to sequences of Latin American and US isolates. The MSP1a repeated sequences of Latin American isolates of A. marginale had nine repeat forms, alpha-phi, which have not been reported previously in North American isolates of A. marginale. Furthermore, the repeated forms tau, sigma and mu were only present in the Brazilian isolates. The results demonstrated that the genetic heterogeneity observed among isolates of A. marginale is common in endemic areas, independent of the predominant tick vector and is consistent with previous studies in which msp4 provided phylogeographic information about A. marginale isolates, while MSP1a was found not to be a useful marker for phylogeographic characterization of A. marginale isolates.     

Prevalence and genetic diversity of Babesia and Anaplasma species in cattle in Sudan

Disease prevalence studies are one of the most valuable tools to demonstrate the risk or impact of certain infections in local and global economies. The data obtained in these studies contribute to develop strategies for disease control. The present study aims to provide information about the prevalence of babesiosis and anaplasmosis in the northern regions of Sudan. Blood samples from four different states of Sudan were collected from apparently healthy cattle (n=692), DNA was extracted and the prevalence of Babesia and Anaplasma species was analyzed by PCR. The results confirmed the presence of Babesia bigemina, Babesia bovis and Anaplasma marginale in cattle in northern Sudan with overall prevalence rates of 4.0%, 1.9% and 6.1%, respectively. Statistical analysis revealed that the prevalence of B. bigemina, B. bovis and A. marginale varies significantly between Sudanese states as well as in different age groups, while gender seems not to have a significant effect on the prevalence of these pathogens among Sudanese cattle. The highest prevalence for B. bigemina was found in the Aljazirah State while the highest number of A. marginale positive samples was reported in River Nile.     

Fatal bovine anaplasmosis in a herd with new genotypes of Anaplasma marginale, Anaplasma ovis and concurrent haemoplasmosis

Haematological and molecular analysis of blood samples was carried out during an outbreak of bovine anaplasmosis in Hungary. Acute disease was observed in five animals, two of which died. Anaplasma-carrier state was diagnosed in 69 (92%) of cattle. Further evaluation of 24 blood samples revealed concurrent infections with Mycoplasma wenyonii and 'CandidatusM. haemobos' in 22 and 21 animals, respectively. In addition, two cows were identified with rickettsaemia. Regarding molecular investigation of potential hard tick vectors, Haemaphysalis inermis and Dermacentor marginatus males collected from the animals were PCR-negative. However, in one pool (out of 18) of Ixodesricinus males, and in six pools (out of 18) of D. reticulatus males the msp4 gene of Anaplasma marginale was detected. In the same I. ricinus pool Anaplasma ovis was also identified. All ticks were negative for haemoplasmas. Anaplasma sequences yielded 97-99% homology to sequences deposited in the Genbank. This is the first report of fatal bovine anaplasmosis associated with divergent A. marginale genotypes and concurrent 'CandidatusM. haemobos' infection, as well as of an A. ovis strain in ticks collected from cattle.     

Molecular identification of Anaplasma marginale and rickettsial endosymbionts in blood-sucking flies (Diptera: Tabanidae, Muscidae) and hard ticks (Acari: Ixodidae)

In an attempt to identify the main vector and possible transmission routes of Anaplasma spp. in a region of Hungary with high prevalence of ovine and bovine anaplasmosis, DNA was extracted from 316 haematophagous arthropods (individually or in pools), including 4 species of ixodid ticks, 6 species of tabanid flies and hornflies. Midichloria-like organisms were identified with PCR (amplifying a portion of the 16S rRNA gene) and sequencing from Dermacentor marginatus and Ixodes ricinus. Significantly higher 16S positive D. marginatus individuals were collected in March than in April, suggesting earlier questing of ticks that contain rickettsial agents (thus endosymbionts). Midichloria- and Wolbachia-like organisms were also found in randomly caught horse flies (Tabanus bovinus and T. tergestinus) as well as hornflies (Haematobia irritans), respectively, with 97-99% similarity to sequences deposited in the GenBank. Although all ticks were negative in the Anaplasma spp.-specific msp4 PCR, four individuals of T. bovinus collected near to grazing cattle were positive for Anaplasma marginale. The results of the present study provide the first molecular evidence for the potential mechanical vector role of T. bovinus in the transmission of A. marginale, and broaden the range of haematophagous arthropods harbouring Midichloria-like bacteria, for the first time in any Dermacentor or Tabanus species.     

Serological Survey of Babesia bovis and Anaplasma marginale in cattle in Tete Province,Mozambique

A serological survey of bovine babesiosis and anaplasmosis in communal cattle was conducted in the northwestern province of Tete, Mozambique. Blood was collected from cattle ranging from 4 to 15 months old from randomly selected farms from six districts. Thirty-nine per cent of all 478 calves tested in Tete Province were seropositive to the ELISA for Babesia bovis antibodies and 63% of all calves were seropositive in the card agglutination test for Anaplasma marginale. Seroprevalence of B. bovis ranged from 22.8% in Tete City District to 48.1% in Angonia District. For A. marginale, it ranged from 34.4% in Angonia District to 87.3% in Moatize District. The dominant factor affecting seroprevalence for both haemoparasites was district and there was a trend for higher intensity of tick control to be associated with a higher seroprevalence of B. bovis and a lower seroprevalence of A. marginale. The obvious differences were the low prevalence of B. bovis in Tete City Council District and the low prevalence of A. marginale in Angonia District. The levels of exposure to B. bovis seen in our study are well below any that could be considered to be consistent with endemic stability, yet they are sufficiently high to ensure that clinical disease would be a risk. The seroprevalence of A. marginale, however, suggests that endemic stability with respect to this disease could exist in districts other than Angonia. There was no strong and consistent relationship between the intensity of control and the likelihood of seropositivity to either of the diseases.     

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 msp1alpha polymerase chain reaction assay for specific detection and differentiation of Anaplasma marginale isolates

Anaplasma marginale is the causative agent of bovine anaplasmosis, a disease which can be protected by vaccination with the less pathogenic Anaplasma species, A. centrale. Currently, there is no polymerase chain reaction (PCR) assay available which differentiates between different species of Anaplasma or which can differentiate isolates of A. marginale within outbreaks and between different countries. A molecular test specific for A. marginale would be ideal for the identification of Anaplasma species in wild ruminants, as possible reservoirs of anaplasmosis, and to differentiate between A. marginale from A. centrale. A PCR assay was designed to amplify the major surface protein 1alpha gene of the rickettsial bovine pathogen, A. marginale both as an inter- and intra-specific test. The test did not amplify A. centrale or A. ovis, and discriminated A. marginale by amplifying repeat regions within the msp1alpha gene which vary in number between many isolates. The nested A. marginale amplicons varied in size from 630 to 1190bp representing one to eight internal repeats. All 22 Australian isolates tested amplified a 630bp product (one repeat) in contrast to all 19 non-Australian isolates tested. Eight sequences from Australian isolates from different geographical regions confirmed the conserved nature of the Australian A. marginale msp1alpha genes. The Australian 'repeat unit' MSP1a deduced amino acid sequence has been designated as Australian type 1. The msp1alpha PCR method developed here enabled the amplification and comparison of A. marginale isolates originating from North and South America, Africa, Israel and Australia. The method is sensitive and specific for A. marginale. Although additional msp1alpha products were amplified from at least two Australian isolates, the results suggest limited introduction of A. marginale into Australia.     

Seroprevalence of Anaplasma marginale in 2 Iowa feedlots and its association with morbidity, mortality, production parameters, and carcass traits

A prospective cohort observational study was conducted to investigate the seroprevalence of Anaplasma marginale in Iowa feedlots and its association with morbidity, mortality, and treatment costs. Blood samples were taken from 659 calves from 31 consigners at processing and classified as seropositive to A. marginale using a competitive enzyme-linked immunosorbent assay (cELISA) with a 30% cutoff. Health and production parameters were modeled by A. marginale serostatus with mixed model regression analysis. The apparent prevalence of seropositive cattle was 15.17% (100/659). When the cELISA positive cutoff was at 42% inhibition, the apparent prevalence was 5.00% (33/659). There was no significant association between A. marginale serostatus and production parameters; however, seropositive status had a weak positive association with undifferentiated fever (P = 0.17). Although prevalence of anaplasmosis in Iowa feedlots is higher than reported in Montana-sourced calves arriving in Canadian feedlots, this was not associated with increased production costs.     

Analysis of world strains of Anaplasma marginale using major surface protein 1a repeat sequences

Anaplasma marginale is a tick-borne pathogen of cattle that causes the disease bovine anaplasmosis worldwide. Major surface proteins (MSPs) are involved in host-pathogen and tick-pathogen interactions and have been used as markers for the genetic characterization of A. marginale strains and phylogenetic studies. MSP1a is involved in the adhesion and transmission of A. marginale by ticks and varies among geographic strains in the number and sequence of amino-terminal tandem repeats. The aim of this study was to characterize the genetic diversity of A. marginale strains collected from countries in North and South America, Europe, Asia, Africa and Australia, inclusive of all continents. In this study, we characterized 131 strains of A. marginale using 79 MSP1a repeat sequences. These results corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. The phylogenetic analyses of MSP1a repeat sequences did not result in clusters according to the geographic origin of A. marginale strains but provided phylogeographic information. Seventy-eight percent of the MSP1a repeat sequences were present in strains from a single geographic region. Strong (> or =80%) support was found for clusters containing sequences from Italian, Spanish, Chinese, Argentinean and South American strains. The phylogenetic analyses of MSP1a repeat sequences suggested tick-pathogen co-evolution and provided evidence of multiple introductions of A. marginale strains from various geographic locations worldwide. These results contribute to the understanding of the genetic diversity and evolution of A. marginale and tick-pathogen interactions.     

Immunization of cattle with Anaplasma marginale derived from tick cell culture.

Anaplasmosis is a hemolytic disease of cattle caused by the ehrlichial tick-borne pathogen Anaplasma marginale. Killed vaccines used for control of anaplasmosis in the US used antigen harvested from infected bovine erythrocytes which was often contaminated with bovine cells and other pathogens. In this study, we performed an initial cattle trial to test A. marginale harvested from tick cell culture as an immunogen for cattle. Eleven yearling Holstein cattle were immunized with the cell culture-derived A. marginale and 11 cattle were non-immunized contact controls. Each vaccine dose contained approximately 2 x 10(10) A. marginale in an oil-based adjuvant. Two immunizations were administered subcutaneously 4 weeks apart and the cattle were challenge-exposed 10 weeks after the second immunization with A. marginale infected blood. Maximum antibody levels as determined by an A. marginale specific competitive ELISA were observed 2 weeks after the last immunization. Antibody responses against major surface proteins (MSPs) 1a and 1beta1 were also characterized and immunized cattle demonstrated a preferential recognition for MSP1beta1. Cattle immunized with the cell culture-derived A. marginale had a significantly lower percent reduction in the packed cell volume (P<0.05) after challenge exposure as compared with the controls and did not display clinical anaplasmosis. The cell culture-derived A. marginale shows promise for use as antigen in development of a new killed vaccine for anaplasmosis.     

Bovine anaplasmosis: susceptibility of seronegative cows from an infected herd to experimental infection with Anaplasma marginale

Adult cows from an Anaplasma marginale-infected herd that were negative to the A marginale rapid card agglutination (RCA) and complement fixation (CF) tests for 1 to 4 years developed acute anaplasmosis after inoculation with 0.5 ml of blood from an A marginale carrier cow. The test cattle were as susceptible as the control cattle of similar ages. Also, 2 cows that had seroconverted from RCA/CF-positive to RCA/CF-negative status naturally were fully susceptible to anaplasmosis when they were experimentally infected. Results of the study indicated that indigenous seronegative cattle in anaplasmosis-enzootic regions probably do not have acquired or natural immunity to A marginale infection.