Identification of Anaplasma ovis appendage-associated protein (AAAP) for development of an indirect ELISA and its application.

BACKGROUND: Ovine anaplasmosis is a tick-borne disease that is caused by Anaplasma ovis in sheep and goats. The pathogen is widely distributed in tropical and subtropical regions of the world. At present, diagnosis of the disease mainly depends on microscopy or nucleic acid based molecular tests, although a few serological tests have been applied for the detection of A. ovis infection. RESULTS: Here we describe the identification of an A. ovis protein that is homologous to the A. marginale appendage-associated protein (AAAP). We expressed a recombinant fragment of this protein for the development of an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of A. ovis. Anaplasma ovis-positive serum showed specific reactivity to recombinantly expressed AAAP (rAAAP), which was further confirmed by the rAAAP ELISA, which also demonstrated no cross-reactivity with sera from animals infected with A. bovis or other related pathogens in sheep and goats. Testing antibody kinetics of five experimentally infected sheep for 1 year demonstrated that the rAAAP ELISA is suitable for the detection of early and persistent infection of A. ovis infections. Investigation of 3138 field-collected serum samples from 54 regions in 23 provinces in China demonstrated that the seroprevalence varied from 9.4% to 65.3%, which is in agreement with previous reports of A. ovis infection. CONCLUSIONS: An A. ovis derived antigenic protein, AAAP, was identified and the antigenicity of the recombinant AAAP was confirmed. Using rAAAP an indirect ELISA assay was established, and the assay has been proven to be an alternative serological diagnostic tool for investigating the prevalence of ovine anaplasmosis of sheep and goats.

Validation of an improved Anaplasma antibody competitive ELISA for detection of Anaplasma ovis antibody in domestic sheep.

An accurate and simple-to-perform new version of a competitive ELISA (cELISA) kit that became commercially available in 2015 for testing of cattle for antibody to Anaplasma marginale was validated for detection of Anaplasma ovis antibody in domestic sheep. True positives and negatives were identified using nested PCR (nPCR) as the gold standard. Negative bovine control sera supplied with the kit were used to calculate % inhibition (%I), designated bovine control ELISA (BcELISA), and this was compared to %I calculated from negative ovine sera derived from hand-raised, pathogen-free sheep, designated ovine control ELISA (OcELISA). The receiver operating characteristics area under the curve was 1.0 with a p value <0.001 regardless of the source of the control sera. The cutoff values for negative BcELISA and OcELISA were <30%I and <27%I, respectively. Our work confirmed that this Anaplasma antibody cELISA kit version 2 can be used with the serum controls supplied in the kit to test for A. ovis antibody in domestic sheep. Furthermore, this work confirmed the historically high infection prevalence (>93%) at the U.S. Sheep Experiment Station (Dubois, Idaho), in spite of efforts to reduce the possibility for iatrogenic transmission there, suggesting high levels of tick-borne transmission.

Evidence of co-infection with Mycobacterium bovis and tick-borne pathogens in a naturally infected sheep flock.

Ticks are responsible for the transmission of pathogens of veterinary importance, including those affecting sheep. The current study was designed to investigate co-infections with tick-borne and other pathogens in a naturally infected sheep flock with poor health condition using serology and PCR. Infection with Anaplasma ovis was detected by serology and PCR in 56% of the animals. The presence of Rickettsia spp. of the Spotted Fever Group (SFG) was detected by PCR and sequence analysis in 31% of the animals. All the animals were negative for Anaplasma phagocytophilum either by serology or PCR. Twelve sheep were randomly selected for anatomopathological studies. Five of these animals presented lesions consistent with Mycobacterium tuberculosis complex (MTBC) infection and spoligotyping confirmed infection with Mycobacterium bovis spoligotype SB0339. Co-infection with tick-borne pathogens and MTBC could contribute to the poor health condition observed in these animals but other uncontrolled factors may also be responsible. The differential expression of immune response genes supported previous findings in ruminants and suggested that infection with tick-borne pathogens and M. bovis may results in unique gene expression patterns in sheep. The results underline the need for further research into the possible role of sheep in the epidemiology of animal tuberculosis.

Serological investigation for West Nile virus, Anaplasma ovis and Leishmania infantum in Greek cattle.

This study investigates the seroprevalence of Greek adult cattle against West Nile virus, Anaplasma ovis and Lehismania infantum. In total, 156 serum samples were examined, drawn from cattle between 2-4 years old. All the examined cattle originated from slaughterhouses of 4 prefectures in Northern Greece (Thessaloniki, Pella, Chalkidiki, Kilkis), in 2 of which (Thessaloniki, Pella) human cases of West Nile virus had been recorded some months before. Thirty out of the 156 (18.6%) samples have tested positive for West Nile virus and fifty-five (35.9%) samples for Anaplasma ovis. All the examined samples tested negative for Leishmania infantum. The prefectures with positive samples against West Nile virus also showed human cases of West Nile virus infections. This should raise questions whether cattle could become markers for West Nile virus activity in high risk areas.

Can Anaplasma ovis in small ruminants be neglected any longer?

Anaplasma species are obligate intracellular rickettsial pathogens transmitted by ticks with an impact on human and animal health. Anaplasma ovis infects sheep and goats in many regions of the world, and it can be diagnosed by different methods like Giemsa staining, PCR or competitive ELISA. In this study, a PCR based on the gene coding for major surface protein 4 (MSP-4) was used to examine field samples collected from sheep in different countries. Altogether, 1161 blood samples from Turkey (n = 830), Iraq (n = 195), Sudan (n = 96) and Portugal (n = 40) were examined, of which 31.4%, 66.6% 41.6% and 82.5%, respectively, were positive. This indicates high prevalence of A. ovis in the countries under investigation, and it can be assumed that the situation in other areas of the world might be similar. Thus, A. ovis should be considered as an important constraint of livestock production, and further efforts are needed to better understand the epidemiology and to implement suitable control measures.

Characterization of pathogen-specific expression of host immune response genes in Anaplasma and Mycobacterium species infected ruminants.

Anaplasma and Mycobacterium species are among the most prevalent bacterial pathogens in European red deer (Cervus elaphus) in south-central Spain and are known to modify gene expression in ruminants. In this study, we used microarray hybridization and real-time RT-PCR analyses to characterize global gene expression profiles in red deer in response to Anaplasma ovis and A. ovis/Mycobacterium bovis/Mycobacterium avium sub. paratuberculosis (MAP) infections, compare the expression of immune response genes between red deer infected with A. ovis, M. bovis and A. ovis/M. bovis/MAP, and characterize the differential expression of immune response genes identified in red deer in cattle infected with M. bovis and Anaplasma marginale. Global gene differential expression in A. ovis- and A. ovis/M. bovis/MAP-infected deer resulted in the modification of common and pathogen-specific cellular biological processes. The differential expression of host immune response genes showed pathogen and host-specific signatures and the effect of infection with multiple pathogens on deer immune response. These results suggested that intracellular bacteria from Anaplasma and Mycobacterium genera produce similar genes expression patterns in infected ruminants. However, pathogen and host-specific differences could contribute to disease diagnosis and treatment in ruminants.

Molecular characterization of two different strains of haemotropic mycoplasmas from a sheep flock with fatal haemolytic anaemia and concomitant Anaplasma ovis infection.

After the first outbreak of fatal Mycoplasma ovis infection (eperythrozoonosis) in a sheep flock in Hungary (1997), a second wave of the disease was noted in 2006, with different seasonal pattern and affected age group, as well as increased mortality (5.5%). The aim of the present study was to molecularly characterize the causative agent and to reveal underlying factors of the second wave of the disease. Remarkably, among the 33 sheep examined, 17 were infected with two strains of haemotropic mycoplasmas. Cloning and sequencing isolates of the latter showed that one of the strains was 99.4-99.8% identical to M. ovis (AF338268), while the second was only 96.8-97.9% identical and contained a 17-bp deletion. Different isolates of both strains were demonstrated in the same animal. When analyzing possible risk factors for fatal disease outcome, we found that among sheep born prior to the 1997 outbreak significantly more animals survived the second outbreak than succumbed to disease. In addition, locally born sheep were less frequently diseased than sheep introduced into the flock from other places. This suggests an immunoprotective effect in some animals. Concurrent infection with Anaplasma ovis was detected in 24 of the 33 evaluated sheep. In conclusion, this is the first study to demonstrate the existence of and characterize two genetically distinct ovine haemotropic mycoplasma strains in a sheep flock with fatal haemolytic anaemia.

Sequence analysis of the msp4 gene of Anaplasma ovis strains.

Anaplasma ovis (Rickettsiales: Anaplasmataceae) is a tick-borne pathogen of sheep, goats and wild ruminants. The genetic diversity of A. ovis strains has not been well characterized due to the lack of sequence information. In this study, we evaluated bighorn sheep (Ovis canadensis) and mule deer (Odocoileus hemionus) from Montana for infection with A. ovis by serology and sequence analysis of the msp4 gene. Antibodies to Anaplasma spp. were detected in 37% and 39% of bighorn sheep and mule deer analyzed, respectively. Four new msp4 genotypes were identified. The A. ovismsp4 sequences identified herein were analyzed together with sequences reported previously for the characterization of the genetic diversity of A. ovis strains in comparison with other Anaplasma spp. The results of these studies demonstrated that although A. ovismsp4 genotypes may vary among geographic regions and between sheep and deer hosts, the variation observed was less than the variation observed between A. marginale and A. phagocytophilum strains. The results reported herein further confirm that A. ovis infection occurs in natural wild ruminant populations in Western United States and that bighorn sheep and mule deer may serve as wildlife reservoirs of A. ovis.

Genetic characterization of Anaplasma ovis strains from bighorn sheep in Montana.

Wildlife reservoir species and genetic diversity of Anaplasma ovis (Rickettsiales: Anaplasmataceae) have been poorly characterized. Bighorn sheep (Ovis canadensis), captured in Montana from December 2004 to January 2005, were tested for antibodies to Anaplasma spp.; the presence of A. ovis was determined by the characterization of major surface protein msp4 sequences. Anaplasma antibodies were detected in 25/180 (14%) sampled bighorn sheep and A. ovis msp4 sequences were amplified by polymerase chain reaction (PCR) and sequenced from 9/23 (39%) of seropositive animals. All animals were negative by PCR for the related pathogens, Anaplasma phagocytophilum and Anaplasma marginale. All msp4 sequences identified in the bighorn sheep were identical and corresponded to a single A. ovis genotype that was identical to a sheep isolate reported previously from Idaho. The finding of a single genotype of A. ovis in this wild herd of bighorn sheep was in contrast to the genetic diversity reported for A. marginale in cattle herds in the western United States and worldwide. These results demonstrated that bighorn sheep may be a wildlife reservoir of A. ovis in Montana.