Longitudinal study of Staphylococcus aureus genotypes isolated from bovine clinical mastitis.

Bovine clinical mastitis is an important problem for the dairy industry, and Staphylococcus aureus is a common mastitis-causing pathogen in many countries. Detailed knowledge on genetic variation of Staph. aureus strains within the bovine population, including changes over time, can be useful for mastitis control programs, because severity of disease and effects on milk production are at least partly strain-associated. Therefore, the major aim of this study was to compare sequence types of Staph. aureus isolated from cases of bovine clinical mastitis from 2002 to 2003 with sequence types of a more recent set of isolates collected from 2013 to 2018, using core genome multi-locus sequence typing (cgMLST). We also wanted to compare antibiotic resistance genes of isolates from the 2 sets, to identify changes that may have occurred over time in the Staph. aureus population. A total of 157 isolates of Staph. aureus, almost equally distributed between the 2 time periods, were subjected to high-throughput sequencing and cgMLST. The results showed that the most prevalent sequence types found among the 2002 to 2003 isolates belonged to the clonal complexes CC97, CC133, and CC151, and that those complexes still dominated among the isolates from 2013 to 2018. However, a population shift from CC133 to CC97 and CC151 over time was observed. Likewise, no important differences in prevalence of antibiotic resistance genes were found between the 2 sets of isolates. As expected, genes belonging to the major facilitator superfamily of transporter proteins, and multidrug and toxic compound extrusion transporters, were very common. Moreover, several genes and mutations conferring resistance to fosfomycin were present, but not in CC97 isolates. The ß-lactamase gene blaZ was found in only 3 out of 81 isolates from 2002 to 2003 and 1 out of 76 isolates in 2013 to 2018. In conclusion, the results indicate that mastitis-associated Staph. aureus strains circulating among dairy cows in Sweden exhibit a remarkable genotypic persistence over a time frame of close to 15 yr.

Staphylococcus aureus genotype B and other genotypes isolated from cow milk in European countries.

Staphylococcus aureus is globally one of the most important pathogens causing contagious mastitis in cattle. Previous studies, however, have demonstrated in Swiss cows that Staph. aureus isolated from bovine intramammary infection is genetically heterogeneous, with Staph. aureus genotype B (GTB) and GTC being the most prominent genotypes. In addition, Staph. aureus GTB was found to be contagious, whereas Staph. aureus GTC and all the remaining genotypes were involved in individual cow disease. The aim of this study was to subtype strains of Staph. aureus isolated from bovine mastitic milk and bulk tank milk to obtain a unified view of the presence of bovine staphylococcal subtypes in 12 European countries. A total of 456 strains of Staph. aureus were subjected to different typing methods: ribosomal spacer PCR, detection of enterotoxin genes, and detection of gene polymorphisms (lukE, coa). Major genotypes with their variants were combined into genotypic clusters (CL). This study revealed 5 major CL representing 76% of all strains and comprised CLB, CLC, CLF, CLI, and CLR. The clusters were characterized by the same genetic properties as the Swiss isolates, demonstrating high clonality of bovine Staph. aureus. Interestingly, CLB was situated in central Europe whereas the other CL were widely disseminated. The remaining 24% of the strains comprised 41 genotypes and variants, some of which (GTAM, GTBG) were restricted to certain countries; many others, however, were observed only once.

Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer.

Staphylococcus aureus is globally one of the most important pathogens causing contagious mastitis in cattle. Previous studies using ribosomal spacer (RS)-PCR, however, demonstrated in Swiss cows that Staph. aureus isolated from bovine intramammary infections are genetically heterogeneous, with Staph. aureus genotype B (GTB) and GTC being the most prominent genotypes. Furthermore, Staph. aureus GTB was found to be contagious, whereas Staph. aureus GTC and all the remaining genotypes were involved in individual cow disease. In addition to RS-PCR, other methods for subtyping Staph. aureus are known, including spa typing and multilocus sequence typing (MLST). They are based on sequencing the spa and various housekeeping genes, respectively. The aim of the present study was to compare the 3 analytic methods using 456 strains of Staph. aureus isolated from milk of bovine intramammary infections and bulk tanks obtained from 12 European countries. Furthermore, the phylogeny of animal Staph. aureus was inferred and the zoonotic transfer of Staph. aureus between cattle and humans was studied. The analyzed strains could be grouped into 6 genotypic clusters, with CLB, CLC, and CLR being the most prominent ones. Comparing the 3 subtyping methods, RS-PCR showed the highest resolution, followed by spa typing and MLST. We found associations among the methods but in many cases they were unsatisfactory except for CLB and CLC. Cluster CLB was positive for clonal complex (CC)8 in 99% of the cases and typically positive for t2953; it is the cattle-adapted form of CC8. Cluster CLC was always positive for tbl 2645 and typically positive for CC705. For CLR and the remaining subtypes, links among the 3 methods were generally poor. Bovine Staph. aureus is highly clonal and a few clones predominate. Animal Staph. aureus always evolve from human strains, such that every human strain may be the ancestor of a novel animal-adapted strain. The zoonotic transfer of IMI- and milk-associated strains of Staph. aureus between cattle and humans seems to be very limited and different hosts are not considered as a source for mutual, spontaneous infections. Spillover events, however, may happen.

Genetic analyses of pathogen-specific mastitis.

The aims of this study were to investigate the presence of genetic variation for susceptibility to pathogen-specific mastitis and to examine whether haplotypes of an identified quantitative trait locus with effect on unspecific mastitis resistance had different effects on specific mastitis pathogens. Bacteriological data on mastitis pathogens were obtained from the diagnostic laboratory at the Swedish National Veterinary Institute. The data were mainly from subclinical cases of mastitis but also clinical cases were included. Variance components were estimated for incidence of the six most frequent pathogens using Markov Chain Monte Carlo methodology via Gibbs sampling. Genetic variation for susceptibility to pathogen-specific mastitis was higher compared to estimates of general resistance to clinical mastitis in most other studies. However, because of the non-random nature of data collection, comparisons to other studies should be made by caution. The effect of haplotype on the risk of being infected by a given mastitis pathogen, relative to other pathogens, was studied using an allele substitution model. Although there were no significant haplotype substitution effects on the resistance to any of the six mastitis pathogens, there was a significant difference between the effects of two of the haplotypes regarding the risk of acquiring a Streptococcus dysgalactiae infection.

An improved method to culture Staphylococcus aureus from bovine milk.

Staphylococcus aureus is an important udder pathogen often associated with subclinical mastitis in dairy cows. Identification of Staph. aureus-positive udder quarters and cows is an important part of control programs to reduce spread of Staph. aureus within and between dairy herds. Therefore, accurate and easy-to-perform culturing methods of Staph. aureus in milk are needed. In the present study, 8 methods for isolation of Staph. aureus in bovine milk samples were investigated. The methods involved different culturing volumes, enrichment, incubation, and freezing processes as well as sedimentation and use of the Mastistrip cassette (SVA, Uppsala, Sweden). Three different sets of milk samples were collected, and 6, 5, and 4 methods were used in each subset of samples. Our results indicate an increased probability of detecting Staph. aureus in milk samples when a simple incubation step (37 degrees C for 18 h) without additives was included before culturing. Using this incubation method, the number of Staph. aureus-positive udder quarters and cows increased by 50 and 29%, respectively, compared with using the standard method of direct culturing of 10 microL of milk. The improved method may be especially useful for detection of low concentrations of Staph. aureus in milk; for example, when screening herds for Staph. aureus.

Identification of potential sources of Staphylococcus aureus in herds with mastitis problems.

Staphylococcus aureus is a common udder pathogen of dairy cows that often causes herd problems. Various mastitis control programs have been used to combat the problem but have not always been efficient in preventing new Staph. aureus infections, indicating the presence of possible sources of infection other than those traditionally considered. Therefore, the aim of the study was to identify potential sources of infection relevant for Staph. aureus mastitis within 5 dairy herds with udder health problems caused by Staph. aureus. Samples were collected from milk of lactating cows, from body sites, and from the environment of lactating cows, dry cows, late pregnant heifers, young heifers 4 to 12 mo old, and heifer calves 0 to 3 mo old. Isolates of Staph. aureus were identified and compared using pulsed-field gel electrophoresis. Four to 7 unique Staph. aureus pulsotypes were found within each herd, with one strain predominating in milk in each herd. The milk pulsotypes were also frequently isolated in body samples, especially on hock skin, and in the immediate environment of lactating cows, and were sometimes found in other animal groups, especially in dry cows and heifer calves 0 to 3 mo old. The prevalence of Staph. aureus in milk and other types of samples varied markedly between herds. Staphylococcus aureus isolates with genotypes indistinguishable from those found in milk also dominated in extra-mammary sites within the dairy herds studied, and hock skin was identified as an important reservoir of Staph. aureus. The results contribute new knowledge necessary to improve strategies for udder health control in herds.

Microbial aetiology of acute clinical mastitis and agent-specific risk factors.

A nation wide study on the microbial aetiology of cases of acute clinical mastitis in Swedish dairy cows was conducted with the aim to investigate changes in the microbial panorama compared to a previous study performed 1994-1995. Another aim was to investigate some agent-specific environmental and individual risk factors. Milk samples were collected from 987 udder quarter cases from 829 cows during six 2-month periods from May 2002 to April 2003, and data on risk factors and demography were collected at sampling by means of a questionnaire. In total, 1056 bacteriological diagnoses were made. The most frequently isolated bacterial species was Staphylococcus aureus constituting 21.3% of the diagnoses, followed by Escherichia coli (15.9%), Streptococcus dysgalactiae (15.6%), Streptococcus uberis (11.1%), coagulase-negative staphylococci (6.2%), Arcanobacterium pyogenes (6.1%) and Klebsiella spp. (4.2%). Samples with no growth or contamination constituted 10.6% and 4.5% of the diagnoses, respectively. A major shift in the panorama of udder pathogens was not observed compared to the survey in 1994-1995. Isolation of Klebsiella spp. was strongly associated with the use of sawdust as bedding material. On the other hand, using sawdust as bedding reduced the risk of isolating S. uberis relative to using straw or peat. The risk of isolating E. coli increased with increasing milk yield and was higher in loose housing systems than in tie stalls. Isolation of S. aureus was associated with tie stalls, and A. pyogenes with low yielding cows and teat lesions. S. dysgalactiae infections were also associated with teat lesions.

Comparison of a commercialized phenotyping system, antimicrobial susceptibility testing, and tuf gene sequence-based genotyping for species-level identification of coagulase-negative staphylococci isolated from cases of bovine mastitis.

In order to evaluate the usefulness of some phenotypic and genotypic methods for species identification of coagulase-negative staphylococci (CNS), isolates were obtained from bovine cases of clinical and sub-clinical mastitis from different geographical areas in Sweden. By using the Staph-Zym test, antimicrobial susceptibility testing, and sequencing of part of the CNS tuf gene and, when needed, part of the 16S rRNA gene we characterized 82 clinical isolates and 24 reference strains of 18 different species of staphylococci. The genotypic methods identified nine different species of CNS among the 82 milk isolates. A comparison with results obtained by tuf gene sequencing showed that Staph-Zym correctly identified CNS reference strains to species level more often than bovine milk CNS isolates (83% and 61%, respectively). In addition, tests supplementary to the Staph-Zym were frequently needed in both groups of isolates (50% of reference strains and 33% of milk isolates) to obtain an identification of the strain. It is notable that Staph-Zym judged two isolates as CNS, although they belonged to other species, could not give a species name in 11% of the bovine CNS isolates, and gave 28% of the isolates an incorrect species name. The present study indicates that the studied phenotypic methods are unreliable for identification of CNS from bovine intra-mammary infections.

Cross-sectional study of the seroprevalence to Borrelia burgdorferi sensu lato and granulocytic Ehrlichia spp. and demographic, clinical and tick-exposure factors in Swedish horses.

A cross-sectional study of the seroprevalence to Borrelia burgdorferi sensu lato and granulocytic Ehrlichia spp. in Swedish horses was conducted to evaluate associations with demographic, clinical and tick-exposure factors. From September 1997-1998, blood samples from 2018 horses were collected from the animals presented to veterinary clinics affiliated with the Swedish Horserace Totalizator Board (regardless of the primary cause for consultation). Standardized questionnaires with information both from owners and attending veterinarians accompanied each blood sample. The apparent seroprevalences to B. burgdorferi s. l. and granulocytic Ehrlichia spp. were 16.8 and 16.7%, respectively. The northern region had the lowest seroprevalences. Four logistic models were developed (controlling for demographic variables). In the disease model of seropositivity to B. burgdorferi s. l., age, breed, geographic region, the serologic titer to granulocytic Ehrlichia spp., season and the diagnosis coffin-joint arthritis were significant. In the tick-exposure model of B. burgdorferi s. l., pasture access the previous year and gender were significant. Age, racing activity, geographic region, season and the serologic titer to B. burgdorferi s. l. were associated with positivity to granulocytic Ehrlichia spp. In the tick-exposure model of granulocytic Ehrlichia spp., pasture access was a risk factor. An interaction between racing activity and geographic region showed that the risk of positive serologic reactions to Ehrlichia spp. was increased in the horse population in the south and middle of Sweden, but only among horses not used for racing. Except for the positive association between coffin-joint arthritis and serologic reactions to B. burgdorferi s. l., there were no significant associations in the multivariable models between non-specific or specific clinical sign or disease with seropositivity to either of these agents.

Effects of the dose of Ehrlichia phagocytophila on the severity of experimental infections in lambs.

Twenty-one lambs were used to investigate whether their response to an infection with Ehrlichia phagocytophila was dose-dependent Four groups of four lambs were infected intravenously with a dilution in physiological saline of E phagocytophila-infected sheep blood containing either 1.3 x 10(5) infected cells, or approximately 43 infected cells, 4.3 infected cells, or 1.3 infected cells (mean values) and four lambs were left uninfected. The incubation period was significantly shorter in the lambs infected with the highest dose of E phagocytophila. However, the clinical and haematological changes observed, and the weekly weight gains of the lambs were independent of the dose of E phagocytophila. As little as one Ephagocytophila infected cell may be enough to transmit the infection.

Detection of granulocytic Ehrlichia species DNA by PCR in persistently infected dogs.

Three female beagle dogs inoculated with granulocytic Ehrlichia species were monitored for four to six months to determine whether there was evidence that the organisms persisted. The dogs were inoculated intravenously with blood containing an Ehrlichia species closely related to Ehrlichia equi and Ehrlichia phagocytophila, and identical to the human granulocytic ehrlichiosis agent with respect to its 16S rRNA gene sequence. The clinical signs were evaluated, and blood samples were collected for haematology, serum biochemistry and serology. Ehrlichial inclusions in the blood were monitored by microscopy, and ehrlichial DNA was detected by the polymerase chain reaction (PCR). Two of the dogs were injected with prednisolone on days 54 to 56 and days 152 to 154 after infection, and the other was injected with prednisolone on days 95 to 97 after infection. The dogs were euthanased and examined postmortem. Ehrlichial inclusions were demonstrated in the neutrophils and seroconversion occurred shortly after inoculation. Two of the dogs developed acute disease with rectal temperatures above 39.0 degrees C, after which no further clinical signs were observed. The administration of corticosteroids seemed to facilitate the detection of ehrlichial inclusions. Ehrlichial DNA was detected intermittently by PCR in blood samples from two of the dogs throughout the study. Persistent infection was demonstrated up to five-and-a-half months after inoculation.

Sero-prevalence of granulocytic Ehrlichia spp. and Borrelia burgdorferi sensu lato in Swedish dogs 1991-94.

In Sweden, 2 tick-borne zoonotic diseases, granulocytic ehrlichiosis and borreliosis, are frequently diagnosed in dogs, using serological assays. The aims of this study were to determine the sero-prevalences of antibodies to Ehrlichia spp. and Borrelia burgdorferi sensu lato during 1991-94 in dogs, not clinically suspected to be infected with either of the 2 agents. Samples (n = 611) were selected from a serum bank using a systematic sampling strategy, stratified across the 4-y period. The stored sera had originally been submitted in order to verify or rule out infection with Sarcoptes scabiei. The overall sero-prevalence for Ehrlichia spp. was 17.7% and for Borrelia burgdorferi sensu lato 3.9% (n = 588). Only a few dogs in the northern part of Sweden were sero-positive for Ehrlichia spp. and none were positive for Borrelia burgdorferi sensu lato. An increased sero-prevalence of Ehrlichia spp. was seen during the years studied. The sero-prevalence of Ehrlichia spp. varied with season. Sero-positivity to both agents increased with age. Both diseases are considered zoonotic, and the increase in sero-prevalence of Ehrlichia spp. over the years may reflect the degree of infection in ticks and may have implications for human health.

A serological and clinical follow-up in horses with confirmed equine granulocytic ehrlichiosis.

For diagnosis of equine granulocytic ehrlichiosis (EGE) serological testing of antibodies to Ehrlichia equi is frequently used. An elevated antibody level is often misinterpreted as confirmative of active infection and results in treatment with antibiotics. If only seropositivity is considered as the diagnostic criterium, many horses showing convalescence titres will be treated. This study was undertaken to obtain information about the kinetics of antibodies during the course of infection and, for this purpose, 45 horses with clinical signs of EGE and confirmed ehrlichiaemia were monitored serologically and clinically over time. For a correct handling of cases with suspected EGE, the following results should be helpful: (i) 44% of the horses in the acute ehrlichiaemic stage were found to be serologically positive to E. equi; (ii) all horses showed a rapid increase in antibody titre, reaching maximum value within a month after the ehrlichiaemic stage; (iii) when 8 months had passed, titres had decreased, but 18 of 24 examined horses were still serologically positive; (iv) after 12-15 months most of the horses (n = 10) were serologically negative; and (v) the period required for complete clinical recovery varied from one day up to 6 months after antibiotic treatment.

[Ehrlichiosis is common among animals but can also occur in humans]. / Ehrlichios vanlig djursjukdom men även människor kan drabbas.

Granulocytic ehrlichiosis is attracting increasing attention as a disease of both humans and animals. Pasture fever in cattle is a form long known in Sweden, and a similar disease was observed in Swedish dogs and horses in the late 1980s. The first cases of human granulocytic ehrlichiosis (HGE) reported from the USA in 1994 proved to have been caused by a species of Erhlichia identical or very similar to the species found in Swedish dogs and horses. Presence of the HGE agent has now been demonstrated in Ixodes ricinus ticks in Sweden. Serological studies among residents in areas of Sweden with a high incidence of ticks have shown the presence of antibodies against the HGE agent in over 10 per cent of cases. To date, there have been about 10 documented cases of HGE in Sweden, a figure to be compared with those for dogs and horses among which ehrlichiosis is relatively common.

Interferon-alpha and haptoglobin in pigs selectively bred for high and low immune response and infected with Mycoplasma hyorhinis.

Pigs selected for high (H) or low (L) combined antibody and cell-mediated immune response were infected with Mycoplasma hyorhinis. Following the infection, arthritis was more severe in the H pigs, while pleuritis and peritonitis were more severe in the L pigs. Since Mycoplasma infections in pigs often cause just mild signs, indicators of the inflammatory response may aid diagnosis of such infections. In addition, data about the genetic influence on inflammatory response indicators are scanty in the pig. The objectives of the study were therefore: firstly, to determine interferon-alpha (IFNalpha) and haptoglobin in M. hyorhinis infected pigs and, secondly, to investigate if the inflammatory response as determined by these indicators was influenced by genetic selection. There was no consistent increase of IFN-alpha in serum following infection. The serum haptoglobin concentration started to increase 3 days post-infection and there was no difference between the two breeding lines. Hence, M. hyorhinis infection in pigs is reflected in increased serum haptoglobin concentration, but no effect of the magnitude of the inflammatory response on this indicator by selection for high or low immune response was observed.

Persistence of Ehrlichia phagocytophila infection in lambs in relation to clinical parameters and antibody responses.

Five lambs were infected experimentally with Ehrlichia phagocytophila and examined regularly during the next six months. The lambs all had recurrences of parasitaemia at various times but had a fever on only 21 per cent of these occasions. A reduced number of leucocytes was observed in all the lambs for at least eight weeks. All the lambs were still infected four months after inoculation with E phagocytophila. After six months, blood from four of the five lambs was infective when inoculated into susceptible lambs.

Trends in the resistance to antimicrobial agents of Streptococcus suis isolates from Denmark and Sweden.

This study was conducted to determine the MIC values of historical and contemporary Streptoccocus suis (serotypes 2 and 7) from Denmark and S. suis (serotype 2) from Sweden. A total of 52 isolates originating from 1967 through 1981 and 156 isolates from 1992 through 1997 in Denmark and 13 isolates from Sweden were examined for their MICs against 20 different antimicrobial agents. Most antimicrobials were active against most isolates. A frequent occurrence of resistance to sulphamethoxazole was observed, with most resistance among historic isolates of serotype 7 and least resistance among isolates from Sweden. A large number of the isolates was resistant to macrolides. However, all historic serotype 2 isolates from Denmark were susceptible, whereas 20.4% of the contemporary isolates were resistant. Among serotype 7 isolates 23.3% of the historic isolates were resistant to macrolides, whereas resistance was found in 44.8% of the contemporary isolates. All isolates from Sweden were susceptible to macrolides. Time-associated frequency of resistance to tetracycline was also found. Only a single historic isolate of serotype 2 was resistant to tetracycline, whereas 43.9% of the contemporary serotype 2 isolates and 15.5% of the contemporary serotype 7 isolates were resistant. Only one (7.7%) of the isolates from Sweden was resistant. The differences in resistance between historic and contemporary isolates from Denmark were statistically significant. This study demonstrated a significant serotype-associated difference in the susceptibility to macrolides and tetracycline and demonstrated that an increase in resistance among S. suis isolates has taken place during the last 15 years to the two most commonly used antimicrobial agents (tylosin and tetracycline) in pig production in Denmark.

Early manifestations of granulocytic ehrlichiosis in dogs inoculated experimentally with a Swedish Ehrlichia species isolate.

Seven beagles were inoculated experimentally with a Swedish canine Ehrlichia species isolate to study its pathogenicity. With respect to the 16S rRNA gene sequence, the isolate was identical to the human granulocytic ehrlichiosis (HGE) agent and closely related to both Ehrlichia equi and E phagocytophila. After an incubation period of four to 11 days, the most prominent clinical signs were high fever for two to five days and depression. All the dogs developed profound thrombocytopenia, moderate leukopenia and a strong serological antibody response. Ehrlichial inclusions were detected in blood neutrophils from four to 14 days after inoculation for four to eight days. Ehrlichial DNA could be detected by polymerase chain reaction during the parasitaemic stage and a few days before and after microscopic inclusions were visible. Postmortem, the dogs showed reactive splenic hyperplasia and non-specific mononuclear reactive hepatitis.

Experimental infection of lambs with an equine granulocytic Ehrlichia species resembling the agent that causes human granulocytic ehrlichiosis (HGE).

Five lambs were inoculated with a granulocytic Ehrlichia species originally isolated from a Swedish horse with granulocytic ehrlichiosis (EGE). The 16S rRNA gene sequence of the Swedish Ehrlichia sp. causing EGE was identical to the sequence of the agent causing human granulocytic ehrlichiosis (HGE). After the inoculation, infected neutrophils and a low serologic response were seen in all lambs, but no clinical symptoms were observed. In one lamb 17% of the neutrophils were infected without a corresponding fever. Six weeks later the lambs were inoculated with an ovine isolate of E. phagocytophila. After challenge with E. phagocytophila the lambs reacted with fever and infected granulocytes. The results presented herein show that the equine Ehrlichia isolate was infective for lambs but generated weak immune response and no distinctive protection from subsequent challenge with E. phagocytophila.

A 16S rRNA-based PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle.

A PCR-based assay was developed for detecting DNA of granulocytic ehrlichiae in blood samples from dogs, horses, and cattle, Primers were designed from 16S rRNA sequence information to specifically amplify DNA from a newly identified Swedish Ehrlichia species. The 16S rRNA nucleotide sequence of this Swedish species differs in only two and three positions from the sequences of Ehrlichia phagocytophila and Ehrlichia equi, respectively, which were also amplified by this PCR system. For evaluation, PCR results were compared with microscopic examination of stained blood smears for the detection of granulocytes containing ehrlichiae (morulae). Thirty-four of 36 microscopically positive samples were also positive by PCR, and 6 microscopically negative samples were negative by PCR as well. Six samples, in which morulae-like structures had been seen, were negative by PCR, also at a lower annealing temperature and when a reamplification of the first PCR products was performed. The identities of the PCR products from some canine and equine isolates were verified by direct DNA sequencing and were found to be identical with the Ehrlichia sequence found in these animal species that had been obtained earlier. The sequences of a segment of approximately 600 nucleotides from two bovine isolates were identical to that of E. phagocytophila, whereas the sequence of another bovine isolate differed in two positions from that of E. phagocytophila and in three positions from the sequences of the canine and equine isolates. Serum samples were analyzed by indirect fluorescent-antibody testing. Seventy-three percent of the animals which were positive by microscopy and PCR also had positive antibody titers. However, it was not possible to rely on a single serological result for diagnosis of present infection. It was, therefore, concluded that PCR was the most reliable method, useful in the clinical laboratory for specific and early diagnosis of granulocytic ehrlichiosis in animals.