Use of dried blood smears for detection of feline hemoplasmas using real-time polymerase chain reaction

The objective of the current study was to determine the sensitivity and specificity of real-time polymerase chain reaction (real-time PCR) for feline hemoplasmas when applied to DNA extracted from dried whole-blood smears in comparison to that for DNA extracted from liquid whole blood. Blood samples were collected into ethylenediamine tetra-acetic acid tubes from 305 cats with possible or suspected hemoplasmosis, and dried blood smears from each sample were prepared. DNA was extracted from blood smears and a 160-microl aliquot of each liquid blood sample by using a robotic extractor and was subjected to real-time PCR for feline glyceraldehyde-3-phosphate dehydrogenase (liquid blood), 18S ribosomal RNA (dried blood), and "Candidatus Mycoplasma haemominutum", Mycoplasma haemofelis, and "Candidatus Mycoplasma turicensis" DNA. When using the results for liquid whole blood as the gold standard, the sensitivity of each assay for "Ca. M. haemominutum", M. haemofelis, and "Ca. M. turicensis" was 49 of 66 (74%), 11 of 13 (85%), and 11 of 20 (55%), respectively. The specificity of each assay was 224 of 234 (96%), 287 of 287 (100%), and 280 of 280 (100%), respectively. When possible, liquid blood samples should be submitted for detection of feline hemoplasmas by using real-time PCR. The improved sensitivity of real-time PCR on blood smears for M. haemofelis compared with that of the other hemoplasma species may reflect the higher organism burdens associated with infection with this species.     

Detection frequency of haemoplasma infections of the domestic cat in Germany

We present epidemiological data on the frequency of infections with haemotrophic Mycoplasma spp. (feline haemoplasmas) in domestic cats in Germany. From November 2004 to October 2006 135 blood samples of anaemic patients and cats without clinical symptoms were examined with conventional and real-time PCR methods. In 15,6 % of the samples DNA of one or more haemoplasma species could be detected. 8,9 % of the samples (12 cats) were infected with "Candidatus Mycoplasma haemominutum, whereas 7,4 % (10 cats) were infected with Mycoplasma haemofelis. Out of these, one cat harboured both species.The recently described species "Candidatus Mycoplasma turicensis" was found in 2.2 % of all samples (3 cats) and was restricted to animals coinfected with M. haemofelis. No correlation could be detected between the infection with haemotrophic Mycoplasma spp. and clinical signs of anaemia or disease. Infections were significantly correlated with age, male gender or coinfections with retroviruses (FIV, FeLV). Our data indicate, that chronically infected carriers without clinical symptoms are frequent in the investigated cat populations in Germany and that the screening of blood-donors for the presence of Mycoplasma spp. infections is advisable before clinical use.     

Prevalence,risk factor analysis,and hematological findings of hemoplasma infection in domestic cats from Valdivia,Southern Chile

Four distinct cat hemoplasma species are recognized worldwide. However, this is the first study to investigate the prevalence, risk factors, and hematological findings of hemoplasmas in cats from Chile. Complete blood count and 16S rRNA real-time PCR for cat hemoplasma species were performed in 384 blood samples from domestic cats in Valdivia, Chile. Among the 384 samples the species-specific prevalence was as follows: ‘Candidatus Mycoplasma haemominutum’ (7.8%), Mycoplasma haemofelis (4.4%), ‘Candidatus Mycoplasma turicensis’ (1%), ‘Ca. M. haemominutum’ + M. haemofelis (0.78%), ‘Ca. M. haemominutum’ + ‘Ca. M. turicensis’ (0.52%), ‘Ca. M. haemominutum’ + ‘Candidatus Mycoplasma haematoparvum’ (0.26%) and ‘Ca. M. haemominutum’ + M. haemofelis + ‘Ca. M. haematoparvum’ (0.26%). Male sex, older age, outdoor access, and FIV status were risk factors for hemoplasmosis. Mycoplasma haemofelis-positive cats had higher mean corpuscular volume and monocyte count. Four hemoplasma species circulate in the cat population of Valdivia. ‘Candidatus M. turicensis’ and ‘Ca. M. haematoparvum’ have been reported for the first time in Chilean cats.     

Prevalences of various hemoplasma species among cats in the United States with possible hemoplasmosis

OBJECTIVE: To determine prevalences of various hemoplasma species among cats in the United States with possible hemoplasmosis and identify risk factors for and clinicopathologic abnormalities associated with infection with each species. DESIGN: Cross-sectional study. Animals-310 cats with cytologic evidence of hemoplasmosis (n = 9) or acute or regenerative anemia (309). PROCEDURES: Blood samples were tested by means of a broad-spectrum conventional PCR assay for hemoplasma DNA and by means of 3 separate species-specific real-time PCR assays for DNA from "Candidatus Mycoplasma haemominutum" (Mhm), Mycoplasma haemofelis (Mhf), and "Candidatus Mycoplasma turicensis" (Mtc). RESULTS: Overall prevalences of Mhm, Mhf, and Mtc infection were 23.2% (72/310), 4.8% (15/310), and 6.5% (20/310), respectively. Mixed infections were detected in 20 (6.5%) cats. Cats infected with hemoplasmas were more likely to be male than were uninfected cats. Infection with FeLV or FIV was significantly associated with infection with Mhf. Compared with uninfected cats, cats infected with Mhf had higher reticulocyte counts, nucleated RBC counts, and mean corpuscular volume; cats infected with Mhm had higher mean corpuscular volume; and cats infected with Mtc had higher monocyte counts. CONCLUSIONS AND CLINICAL RELEVANCE: Results supported the suggestion that these 3 hemoplasma species commonly occur among cats in the United States and that pathogenicity of the 3 species varies.     

The prevalence of Bartonella, hemoplasma, and Rickettsia felis infections in domestic cats and in cat fleas in Ontario

The prevalence of persistent bacteremic Bartonella spp. and hemoplasma infections was determined in healthy pet cats in Ontario. Blood samples from healthy cats sent to a diagnostic laboratory for routine health assessment over the course of 1 y were tested for Bartonella spp. using both polymerase chain reaction (PCR) and blood culture, and for the presence of hemoplasma by PCR. The overall prevalence of Bartonella spp. by PCR and by culture combined was 4.3% (28/646) [3.7% (24/646) Bartonella henselae, 0.6% (4/646) Bartonella clarridgeiae]. The novel B. henselae PCR developed for this study demonstrated nearly twice the sensitivity of bacterial isolation. The overall prevalence of hemoplasma was 4% (30/742) [3.3% (25/742) Candidatus Mycoplasma haemominutum, 0.7% (5/742) Mycoplasma haemofelis]. There was no significant difference between the prevalence of infection by season or by age (≤ 2 y, > 2 y). Candidatus Mycoplasma turicensis was identified, for the first time in Canada, in 1 cat. The prevalence of Bartonella (58%) and hemoplasma (47% M. haemofelis, 13% M. haemominutum) in blood from a small sampling (n = 45) of stray cats was considerably higher than that found in healthy pet cats.     

Efficacy of oral supplementation with L-lysine in cats latently infected with feline herpesvirus

OBJECTIVE: To examine the effects of orally administered L-lysine on clinical signs of feline herpesvirus type 1 (FHV-1) infection and ocular shedding of FHV-1 in latently infected cats. ANIMALS: 14 young adult, FHV-1-naive cats. PROCEDURE: Five months after primary conjunctival inoculation with FHV-1, cats were rehoused and assigned to receive 400 mg of L-lysine in food once daily for 30 days or food only. On day 15, all cats received methylprednisolone to induce viral reactivation. Clinical signs of infection were graded, and viral shedding was assessed by a polymerase chain reaction assay throughout our study. Peak and trough plasma amino acid concentrations were assessed on day 30. RESULTS: Fewer cats and eyes were affected by conjunctivitis, and onset of clinical signs of infection was delayed on average by 7 days in cats receiving L-lysine, compared with cats in the control group; however, significant differences between groups were not demonstrated. Significantly fewer viral shedding episodes were identified in the treatment group cats, compared with the control group cats, after rehousing but not following corticosteroid-induced viral reactivation. Mean plasma L-lysine concentration was significantly increased at 3 hours but not at 24 hours after L-lysine administration. Plasma arginine concentration was not significantly altered. CONCLUSIONS AND CLINICAL RELEVANCE: Once daily oral administration of 400 mg of L-lysine to cats latently infected with FHV-1 was associated with reduced viral shedding following changes in housing and husbandry but not following corticosteroid administration. This dose caused a significant but short-term increase in plasma L-lysine concentration without altering plasma arginine concentration or inducing adverse clinical effects.     

"Candidatus Mycoplasma haemominutum" infections in 21 client-owned cats

Medical records were reviewed for 21 clinically ill cats testing positive for deoxyribonucleic acid (DNA) of "Candidatus Mycoplasma haemominutum" in their blood. Fever, anorexia, lethargy, and anemia were among the most common abnormalities recorded. Thirteen cats were anemic; seven had evidence of other diseases that could have been the primary cause of anemia or activated hemoplasmosis. For six cats, "Candidatus Mycoplasma haemominutum" was the only recognizable cause of the anemia. Of these cats, anemia resolved in one cat without treatment and in three cats that were treated with doxycycline, with or without prednisone. Results of the study suggest that this hemoplasma species can be a primary pathogen in cats.     

Design,optimization, and application of a conventional PCR assay with an internal control for detection of ‘Candidatus Mycoplasma turicensis’ 16S rDNA in domestic cats from Brazil

Background: ‘Candidatus Mycoplasma turicensis’ (CMtc) is a hemotrophic bacterial species that can, alone or in combination, induce anemia in cats. The diagnostic test of choice for hemoplasma infections is PCR. Conventional PCR assays have been developed for the detection of Mycoplasma haemofelis (Mhf) and ‘Candidatus M. haemominutum’ (CMhm) but not for CMtc. Although real‐time PCR assays have been reported for all of the feline hemoplasmas, the expense of necessary instrumentation precludes its use in Brazil and many other countries. Objectives: The goals of this study were to develop and optimize a conventional PCR assay to diagnose CMtc using an internal control to detect false‐negative results, and to evaluate the occurrence of CMtc infection in domestic cats from Brazil. Methods: Species‐specific primers were designed and a PCR assay was developed for the detection of CMtc 16S rDNA in cat blood. Sensitivity was determined by serial 10‐fold dilutions of plasmid and DNA extracted from blood from an experimentally infected cat. EDTA blood samples from 373 cats were collected. DNA was extracted using a silica‐based protocol and tested using the PCR assay. Results: Primer concentration, annealing temperature, and MgCl₂ concentration were optimized in the presence and absence of the internal control. Two samples negative for the internal control were excluded. Of the remaining 371 samples (117 healthy and 254 unhealthy cats), 17 (4.6%) were positive for CMtc. Conclusion: These results demonstrate the utility of an optimized PCR assay to detect CMtc in feline blood samples. We also report for the first time the prevalence of CMtc infection in domestic cats in Brazil.     

Feline hemotropic mycoplasmosis (feline hemobartonellosis).

Hemotropic mycoplasmas represent an important cause of anemia in cats worldwide. Previously known as Haemobartonella species, sequencing of the 16S rRNA genes of these organisms has led to their reclassification as mycoplasmas. Two species have been identified in cats, M haemofelis and "Candidatus M haemominutum." The latter organism alone has not been associated with disease in naturally infected cats but may cause anemia in FeLV-infected cats and accelerate development of FeLV-induced myeloproliferative disease. The mode of transmission of these organisms remains enigmatic. Nevertheless, development of sensitive DNA-based tests for these unculturable organisms has improved the understanding of the epidemiology and pathogenesis of FHM. Cats with clinical signs and laboratory abnormalities consistent with FHM should be treated with doxycycline; enrofloxacin may represent an effective alternative. Transfusion with packed red blood cells after cross-matching may be required for severely anemia cats, and addition of prednisone may be required if the diagnosis of FHM is uncertain, or response to antimicrobials alone is insufficient. Affected cats should be tested for FeLV, the most common concurrent infection in cats with FHM.     

Detection and molecular characterization of feline hemoplasmas in wild felid species in Iran in the Middle East

Three feline hemoplasma species exist in felids: Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’, and ‘Candidatus Mycoplasma turicensis’.The aims of the study were to determine the presence of, and molecularly characterize, any hemoplasmas in wild felids, including the endangered Persian leopard in Iran, the Middle East.Blood samples were collected from 19 wild felids, including three Persian leopards. Using species-specific hemoplasma PCRs and ELISA serological testing for feline leukaemia virus and feline immunodeficiency virus (FIV), two Persian leopards were found to be infected with ‘Ca. M. haemominutum’ and were seropositive for FIV. Partial 16S rRNA gene sequences were generated for these ‘Ca. M. haemominutum’ species and subsequent phylogenetic analysis revealed 97.70% to 99.45% sequence identity with those found in domestic cats from Iran and other countries.This study confirms the presence of ‘Ca. M. haemominutum’ and concurrent FIV antibody in wild felids in Iran. This represents the first report of hemoplasma in wild felids in the Middle East as well as the first report of infection in Persian leopards.     

Lack of cross-protection against Mycoplasma haemofelis infection and signs of enhancement in “Candidatus Mycoplasma turicensis”-recovered cats

“Mycoplasma haemofelis” and “Candidatus Mycoplasma turicensis” are feline hemoplasmas that induce hemolytic anemia. Protection from homologous re-challenge was recently demonstrated in cats recovered from primary infection. Here, we determined if cats recovered from “Cand. M. turicensis” infection were protected against infections with the more pathogenic M. haemofelis. Ten specified pathogen-free cats were exposed to M. haemofelis. Five of the ten cats had recovered from “Cand. M. turicensis” bacteremia (group A), and five cats were naïve controls (group B). No cross-protection was observed. By contrast, the “Cand. M. turicensis”-recovered cats displayed faster M. haemofelis infection onset (earlier PCR-positive and anemic) than the controls. No “Cand. M. turicensis” was detected in any cat. M. haemofelis shedding was observed in saliva, feces and urine. In both groups, evidence of a Th1 response was observed (high IFN-γ, low IL-4), but IL-10 levels were also high. In group A, total, CD4+ and CD8+ T cells increased within days after M. haemofelis exposure. At times of maximal bacteremia, macrocytic hypochromic anemia, neutropenia, monocytosis and a decrease in leukocyte, eosinophil, and lymphocyte counts and subsets thereof (B- and T-cells, CD4+, CD8+ and CD4+CD25+ cells) were particularly significant in group A. Moreover, an increase in protein concentrations, hypoalbuminemia and a polyclonal hypergammaglobulinemia were observed. Five of ten M. haemofelis-infected cats subsequently cleared bacteremia without antibiotic treatment. In conclusion, the study suggests that a previous hemoplasma infection, even when the cat has ostensibly recovered, may influence subsequent infections, lead to an enhancement phenomenon and other differences in infection kinetics.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0240-x) contains supplementary material, which is available to authorized users.     

Immune-mediated erythroid and megakaryocytic aplasia in a cat

CASE DESCRIPTION: A 6-month-old domestic shorthair cat was evaluated because of acute lethargy. CLINICAL FINDINGS: Severe nonregenerative anemia and thrombocytopenia were identified. Cytologic examination of a bone marrow aspirate revealed selective erythroid and mega-karyocytic aplasia and a high number of apparently normal small lymphocytes. Infectious agents implicated in feline hematologic disorders were excluded on the basis of serologic tests or PCR amplification, including FeLV, Ehrlichia canis, Anaplasma phagocytophilum, Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum, and Candidatus Myco-plasma turicensis. TREATMENT AND OUTCOME: A 10-day course of prednisolone administration did not improve the hematologic disorder. Administration of human polyclonal immunoglobulins preceded increased reticulocyte count by 3 days. A second bone marrow examination confirmed restoration of erythroblasts and megakaryocytes. After 1 relapse, the disease was successfully controlled with prednisolone for > 3 years. CLINICAL RELEVANCE: Immune-mediated bone marrow aplasia is rare in cats and usually affects only erythrocyte progenitors. Concomitant involvement of erythroid and megakaryocytic cell lines can be successfully treated via immunosuppressive therapy. Human immunoglobulins seem to be well tolerated in cats; however, proof of a beneficial effect requires further study.     

Hemoplasmas in wild canids and felids in Brazil

Hemotropic mycoplasmas, epicellular erythrocytic bacterial parasites lacking a cell wall, are the causative agents of infectious anemia in numerous mammalian species. The presence of hemotropic mycoplasmas in blood samples of neotropical and exotic wild canids and felids from Brazilian zoos were recorded using molecular techniques. Blood samples were collected from 146 Brazilian wild felids, 19 exotic felids, 3 European wolves (Canis lupus), and from 97 Brazilian wild canids from zoos in the Brazilian states of S?o Paulo and Mato Grosso and the Federal District. Using conventional polymerase chain reaction (PCR), this work found 22 (13%) wild felids positive to Candidatus Mycoplasma haemominutum [4 jaguars (Panthera onca); 3 pumas (Puma concolor); 10 ocelots (Leopardus pardalis); 2 jaguarondis (Puma yagouaroundi); and 3 little spotted cats (Leopardus tigrinus)]. Only one little spotted cat (Leopardus tigrinus) was positive to Mycoplasma haemofelis, and none was positive to Candidatus Mycoplasma turicensis. Two bush dogs (Speothos venaticus) were positive for a Mycoplasma sp. closely related to Candidatus Mycoplasma haematoparvum, and two European wolves were positive for a Mycoplasma sp. closely related to Candidatus Mycoplasma haemominutum. This is the first study regarding the molecular detection of hemotropic mycoplasmas in wild canids.     

Prevalence of hemoplasmas and Bartonella species in client-owned cats in Beijing and Shanghai,China

A year-round molecular epidemiological survey (2017 to 2018) was conducted on three hemoplasmas and two Bartonella species with zoonotic potential in client-owned cats in Beijing and Shanghai. Among 668 specimens, the overall hemoplasma-positive rate was 4.9% (3.4% for Candidatus Mycoplasma haemominutum, 0.9% for Mycoplasma haemofelis and 1.2% for Candidatus Mycoplasma turicensis). The overall Bartonella-positive rate was 8.5% (4.8% for B. henselae and 4.3% for B. clarridgeiae). Age, breed, ectoparasiticide use and stray history, but not city, season and gender, were significantly associated with the positive rates of one or more pathogens. This is also the first report on the prevalence of Candidatus Mycoplasma turicensis in cats in China.     

Detection of mixed infections with "Candidatus Mycoplasma haemominutum" and Mycoplasma haemofelis using real-time TaqMan polymerase chain reaction.

The objective of this study was to evaluate the use of real-time TaqMan PCR assays for detection of coinfections with "Candidatus Mycoplasma haemominutum" (Mhm), and Mycoplasma haemofelis (Mhf), in vitro and over time in experimentally infected cats. First, the ability of each real-time PCR assay to detect and quantify mixed infections was determined in vitro by testing mixtures of plasmids containing Mhm and Mhf 16S rDNA with each assay. Subsequently, 4 specific pathogen-free (SPF) cats, 2 of which were splenectomized, were inoculated with blood from a cat infected with both Mhm and Mhf. Sixteen blood samples were then collected from each cat over a 55-day period. Each of the 64 postinoculation samples was tested using both conventional polymerase chain reaction (cPCR) and real-time PCR for the 16S rRNA gene of each organism. When applied to mixtures of plasmid DNA from each species, the results of quantitation with each of the real-time PCR assays approximately reflected the number of plasmid copies present. Forty-nine of 64 post-inoculation samples (77%) were positive using both cPCR and real-time PCR, 4 (6%) were positive using cPCR only, and 3 (5%) were positive using real-time PCR only. Both organisms were detected in 23 samples using real-time PCR. Mixed infections were not detected using cPCR. The size of the corresponding cPCR products suggested infection with Mhm in 4 and Mhf in 18 of these samples. The use of multiple separate real-time PCR assays rather than cPCR alone should thus be considered for epidemiologic studies of hemoplasmosis in cats.     

Prevalence of Bartonella species,Rickettsia felis,haemoplasmas and the Ehrlichia group in the blood of cats and fleas in eastern Australia

Objectives To define the prevalence of Bartonella spp., Rickettsia felis, Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’ (Mhm) and ‘Candidatus Mycoplasma turicensis’ (Mtc) in cats and their fleas in eastern Australia. Design and procedure Conventional PCR assays that detect Bartonella spp., M. haemofelis, Mhm, Mtc, Rickettsia spp., Ehrlichia spp., Anaplasma spp. and Neorickettsia spp. were performed on DNA extracted from blood and fleas collected from 111 cats. Cat sera were assayed by ELISA for IgG of Bartonella spp. Results DNA of M. haemofelis, Mtc and Mhm was amplified from 1 (0.9%), 1 (0.9%) and 17 cats (15.3%), respectively. Only DNA of Mhm was amplified from the 62 of 111 pooled flea samples (flea sets; 55.9%). Overall, the prevalence rates for Bartonella spp. DNA in the cats and the flea sets was 16.2% (18 cats) and 28.8% (32 flea sets), respectively. Bartonella spp. IgG was detected in 42 cats (37.8%), of which 11 (26.2%) were positive for Bartonella spp. DNA in their blood. R. felis DNA was amplified from 22 flea sets (19.8%), but not from cats. Overall, DNA of one or more of the organisms was amplified from 27% (30) of cats and 67.6% (75) of the flea sets. Conclusions This is the first Australian study to determine the prevalence of R. felis and B. clarridgeiae in both fleas and the cats from which they were collected. Flea-associated infectious agents are common in cats and fleas in eastern Australia and support the recommendation that stringent flea control be maintained on cats.     

Molecular detection of Bartonella henselae and Bartonella clarridgeiae in clinical samples of pet cats from Southern Italy

Bartonella henselae is considered an emerging pathogen of veterinary and medical interest that can be occasionally transmitted to humans. Cats are considered to be the only reservoir host for B. henselae. In this study, we used a nested-PCR assay to investigate the prevalence of B. henselae and Bartonella clarridgeiae DNA in peripheral blood samples, fine needle lymph node aspirate specimens and oral swabs from 85 cats in order to develop an easy diagnostic strategy for the selection of infection-free cats that are being considered as pets, especially for immunocompromised patients. Overall, molecular analysis showed that 71 cats (83.5%) tested PCR positive for the presence of B. henselae DNA. PCR amplification of DNA B. henselae produced positive products from lymph node aspirate specimens (62/85; 72.9%) similar to those obtained from blood samples (60/85; 70.6%) and higher than those from oral swabs (51/85; 60%) of cats. No PCR product was obtained for B. clarridgeiae. The simultaneous analysis of three different clinical samples in our study increased the diagnostic possibilities for B. henselae infection in the examined cats from 60–72.9% to 83.5%. Lymph node aspirates were found to be the most effective clinical samples for the detection of B. henselae and blood samples were the next best. Oral swab samples were used in this study with good results when considered in combination with blood and/or lymph node aspiration. The use of nested-PCR assay on these three clinical samples may enhance the diagnostic sensitivity for bartonellosis in cats irrespective of the clinical status of animals.     

Phylogenetic analysis of Mycoplasma suis isolates based on 16S rRNA gene sequence in China

To perform phylogenetic analysis of Mycoplasma suis isolates derived from China to define the nature of this pathogen, nearly complete of 16S rRNA genes from Chongqing, Sichuan, Henan and Guangdong isolates were amplified by PCR and sequenced. The four sequences from the blood samples in this study, with other 17 Hemoplasmas sequences and related 3 mycoplasma sequences available in the GenBank, were aligned using Clustal X (version 1.83) sequences alignment program. Maximum parsimony, neighbor-joining and minimum evolution (MEGA 4.0) algorithms were used to create phylogenetic trees. Phylogenetic analysis of these sequences showed that all hemoplasma species were located within a single clade and were most closely related to M. pneumoniae group. The hemoplasma species were further subdivided into two distinct groups, one containing M.wenyonii, M.suis and Candidatus M. haemominutum and the other containing M. haemofelis and M. haemocanis. Within the former clade, four M.suis isolates from Mainland China and other M.suis species formed a monophyletic group in the tree. A tendency of clear geographical grouping of the isolate was evident.     

Superinfection occurs in Anaplasma phagocytophilum infected sheep irrespective of infection phase and protection status

Background

Anaplasma phagocytophilum infection in domestic ruminants is widespread in the coastal areas of southern Norway. The bacteria may persist in mammalian hosts. Several genetic variants of A. phagocytophilum exist. In the present study, we investigate whether superinfection occurs in the acute and persistent phase of the infection.

Methods

Five-month-old lambs of the Norwegian Dala breed were experimentally infected with two 16S rRNA gene variants of A. phagocytophilum, i.e. A. phagocytophilum variant 1 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"M73220","term_id":"148293","term_text":"M73220"}}M73220) and variant 2 (GenBank acc. no. {"type":"entrez-nucleotide","attrs":{"text":"AF336220","term_id":"13325085","term_text":"AF336220"}}AF336220). Eighteen lambs were used, two lambs in each group. Eight groups were experimentally inoculated with either variant 1 or 2 on day 0. Six of these groups were then challenged with the other variant on either days 7, 42 or 84, respectively. One group was left uninfected. The occurrence of A. phagocytophilum in blood samples was determined using semi-nested PCR analysis and gene sequencing. Specific antibodies were measured by an indirect immunofluorescence antibody assay (IFA).

Results

A. phagocytophilum variant 1 and 2 differed significantly with regards to clinical reaction and cross-immunity in infected lambs. Both variants were found in the blood after challenge. However, variant 1 was detected most frequently.

Conclusion

The present experiment indicates that superinfection of different genotypes occurs during the acute as well as the persistent phase of an A. phagocytophilum infection, even in lambs protected against the challenged infection.