Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum.

BACKGROUND: Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain. RESULTS: Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S. CONCLUSIONS: Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum.

Association between ecological factors and the presence of Rhipicephalus (Boophilus) microplus larvae in Puerto Rico.

A prevalence study was conducted to survey tick larvae populations in Puerto Rico (PR), compare the number of infested sites with Rhipicephalus (Boophilus) microplus larvae between the wet and dry season, and assess the associations of ecologic factors on the presence of R. microplus larvae. Ninety-six sites were selected using a GIS-based sampling method. Each site was sampled twice; the first sampling was performed during the dry season (March 4-18, 2007) and the second sampling during the wet season (August 13-26, 2007). Sites were sampled using a tick drag with a 1-m(2) white flannel cloth along a 50-m straight course. Only 2 tick species were identified. In the dry season, 15 sites (0.16, 95 % CI = 0.09-0.24) were identified with R. microplus larvae (n = 606) and 9 sites (0.09, 95 % CI = 0.04-0.17) with Dermacentor (Anocentor) nitens larvae (n = 779), whereas in the wet season 5 sites (0.05, 95 % CI = 0.02-0.12) were identified with R. microplus (n = 94), and 5 sites (0.05 %, 95 % CI = 0.02-0.12) with D. nitens (n = 275). Difference in the number of infested sites with R. microplus was significant (P = 0.031) between the 2 seasons. Factors associated with the presence of R. microplus larvae in PR were wind speed of >4.0 km/h (OR = 0.07, 95 % CI = 0.01-0.63), more than 25 % bushes and shrubs on the site (OR = 11, 95 % CI = 1.6-71), and presence of cattle on the site (OR = 26, 95 % CI = 3.4-188).

Diagnostic accuracy of methods for detecting Anaplasma marginale infection in lactating dairy cattle of Puerto Rico.

Bovine anaplasmosis (BA) is a hemoparasitic disease of great importance in cattle within the tropical and subtropical regions of the world. Control programs for BA require accurate diagnostic assays but validation can be challenging because the true disease status of all animals is frequently not known with certainty. The objective of this study was to estimate the accuracy of assays for detection of Anaplasma marginale infection in lactating dairy cattle of Puerto Rico using Bayesian methods without a perfect reference test. There were 2,331 cattle with complete diagnostic results sampled from 79 herds, and the prevalence of BA was estimated as 22% (95% probability interval [PI]: 19-25%). The sensitivity (Se) and specificity (Sp) of a major surface protein 5 competitive enzyme-linked immunosorbent assay (MSP-5 cELISA) were estimated as 99% (95% PI: 96-100%) and 89% (95% PI: 87-92%), respectively. The Se and Sp of a quantitative polymerase chain reaction (qPCR) were 67% (95% PI: 60-74%) and 99% (95% PI: 99-100%). The Se and Sp of a card agglutination test were 34% (95% PI: 29-39%) and 99% (95% PI: 99-100%). Area under the receiver-operating characteristic curve for the MSP-5 cELISA was 0.748 (95% PI: 0.71-0.79). The MSP-5 cELISA appears to be the test of choice for screening cattle for subclinical BA based on the high estimated Se, rapidity of results, relative low cost, and ease of standardization.

Performance of a commercially available in-clinic ELISA for the detection of antibodies against Anaplasma phagocytophilum, Ehrlichia canis, and Borrelia burgdorferi and Dirofilaria immitis antigen in dogs.

OBJECTIVE: To evaluate the sensitivity and specificity of a commercially available in-clinic ELISA for detection of heartworm infection and tick-borne diseases in dogs. SAMPLE POPULATION: 846 serum, plasma, or blood samples obtained from dogs. PROCEDURES: Samples were evaluated via the in-clinic ELISA to detect antibodies against Anaplasma phagocytophilum, Ehrlichia canis, and Borrelia burgdorferi and Dirofilaria immitis (heartworm) antigen. True infection or immunologic status of samples was assessed by use of results of necropsy, an antigen assay for D immitis, and immunofluorescence assay or western blot analysis for antibodies against B burgdorferi, E canis, and A phagocytophilum. RESULTS: Sensitivity and specificity of the in-clinic ELISA for detection of heartworm antigen (99.2% and 100%, respectively), antibodies against B burgdorferi (98.8% and 100%, respectively), and antibodies against E canis (96.2% and 100%, respectively) were similar to results for a similar commercial ELISA. In samples obtained from dogs in the northeast and upper Midwest of the United States, sensitivity and specificity of the in-clinic ELISA for antibodies against Anaplasma spp were 99.1% and 100%, respectively, compared with results for an immunofluorescence assay. Samples from 2 dogs experimentally infected with the NY18 strain of A phagocytophilum were tested by use of the in-clinic ELISA, and antibodies against A phagocytophilum were detected by 8 days after inoculation. Antibodies against Anaplasma platys in experimentally infected dogs cross-reacted with the A phagocytophilum analyte. Coinfections were identified in several of the canine serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: The commercially available in-clinic ELISA could be used by veterinarians to screen dogs for heartworm infection and for exposure to tick-borne pathogens.

Diversity of Anaplasma phagocytophilum strains, USA.

We analyzed the structure of the expression site encoding the immunoprotective protein MSP2/P44 from multiple Anaplasma phagocytophilum strains in the United States. The sequence of p44ESup1 had diverged in Ap-variant 1 strains infecting ruminants. In contrast, no differences were detected between A. phagocytophilum strains infecting humans and domestic dogs.

Comparison of blood values in foraging, nesting, and stranded loggerhead turtles (Caretta caretta) along the coast of Georgia, USA.

The health status of 83 loggerhead sea turtles (Caretta caretta; 39 foraging, 31 nesting, and 13 stranded turtles) was analyzed using physical examinations, hematology, plasma biochemistry, plasma protein electrophoresis, and toxicologic parameters. Significant differences were noted in a number of health parameters between turtles exhibiting each of these behaviors. On physical examinations, stranded turtles had the highest prevalence of heavy carapace epibiont loads, miscellaneous abnormalities, emaciation, and weakness. Differences in hematologic values included a lower packed cell volume, higher number of lymphocytes, and lower number of monocytes in stranded turtles; lower white blood cell counts in foraging turtles; and significant differences in total solid values among turtles exhibiting all behaviors with the lowest values in stranded turtles and the highest values in nesting turtles. Differences in plasma biochemistry values included the highest uric acid, creatine kinase, and CO(2) values in stranded turtles; the highest glucose and potassium values in foraging turtles; and the highest cholesterol and triglyceride values, and lowest alanine aminotransferase, in nesting turtles. Differences in total protein, albumin, and globulin were found using plasma biochemistry values, with lowest values in stranded turtles and highest values in nesting females, whereas differences in blood urea nitrogen between turtles included the lowest values in nesting turtles and the highest in foraging turtles. Plasma organochlorine and polychlorinated biphenyl levels were below their limits of quantification in the 39 foraging, 11 nesting, and three stranded turtles tested. A statistically significant difference was noted in the level of whole blood mercury between the 23 foraging and 12 nesting turtles tested. There was no difference in arsenic or lead levels between turtles exhibiting any of the three behaviors. Although a few limitations exist with the present study and include unknown ambient temperatures, turtle handling times that varied from 15 min to 53 min per turtle, and the use of a different laboratory for processing complete blood counts and plasma biochemistries in stranded versus foraging and nesting turtles, we provide baseline blood values for two cohorts (foraging and nesting) of loggerhead sea turtles on the coast of Georgia. Additionally, we demonstrate significant differences in clinical findings and blood parameters between foraging, nesting, and stranded loggerhead turtles in the region.

Urinalysis in Asian elephants (Elephas maximus).

Urine was collected from 22 healthy female adult Asian elephants (Elephas maximus) and analyzed for the purpose of determining normal biochemical and microscopic parameters. Findings included urine that was less concentrated compared to other mammals, predominantly alkaline pH, crystalluria of varying types in all samples, and minimal cellularity. Glucose and urobilinogen were not detected in any samples. Trace ketones and trace bilirubin occurred in two different samples. Trace blood was identified in another sample. Three samples tested positive for protein via dipstick but were confirmed negative through the sulfosalicylic acid test. Two samples contained mucus threads. Bacteria were seen microscopically in four samples, and could be cultured from six others, but, because of the lack of an associated inflammatory response and the heterogeneous populations of organisms observed, were considered to be contaminants from the distal urethra, the vestibulovulva, or the environment. Because of the variability in elephant urine, baseline values for elephants within captive herds should be obtained and regular assessments should be performed over time to allow trending of data. Establishment of normal urine values provides an important tool in elephant health care.

Cytologic and immunohistochemical characterization of a lung carcinoid in a dog.

An 11-year-old neutered male Yorkshire Terrier was presented to the Haemaru Referral Animal Hospital with a history of unresponsive tracheal collapse and an incidental finding of a lung nodule in the left caudal lung lobe on radiography. Thorough physical examination and imaging studies revealed no other masses. Cytologic examination of C-arm mobile fluoroscopy-guided fine-needle aspirates revealed numerous free nuclei and a low number of small round cells with moderate to abundant pale basophilic cytoplasm. Some cells contained indistinct basophilic granules in their cytoplasm, and extracellular pink material was noted. A caudal lung lobectomy was performed, and histologic evaluation of the mass revealed round to polygonal cells with abundant eosinophilic granular cytoplasm and round nuclei with mild anisokaryosis and 0-3 mitotic figures per high-power field. Cells were arranged in packets separated by fine fibrovascular stroma, suggestive of a pulmonary neuroendocrine neoplasm, specifically a carcinoma/carcinoid. The cells were immunoreactive for chromogranin A and neuron-specific enolase, and negative for cytokeratin, synaptophysin, calcitonin, thyroglobulin, parathyroid hormone, CD79a, light lambda, and vimentin. With these findings the tumor was diagnosed as a primary lung carcinoid. Eleven months after resection, there was no evidence of tumor regrowth or metastasis. The absence of necrosis, few mitotic figures, minimal pleomorphism, and benign behavior of this tumor resembled those of a typical carcinoid in humans.

Systemic infection with Geomyces organisms in a dog with lytic bone lesions.

CASE DESCRIPTION: A 5-year-old neutered male mixed-breed dog was evaluated by a veterinarian because of a 4-week history of progressive lethargy and poor appetite; the dog was then examined at a referral hospital. CLINICAL FINDINGS: Hyperglobulinemia was identified via serum biochemical analyses performed before and after arrival at the hospital. Lysis of sternebrae 1 and 2 and sternal lymphadenopathy were detected radiographically. Fine-needle aspirates were collected from the affected sternebrae and lymph node for cytologic examination; findings were consistent with pyogranulomatous inflammation associated with fungal infiltrates. Geomyces organisms were identified via microbial culture of sternebral aspirates. TREATMENT AND OUTCOME: Treatment consisted of oral administration of itraconazole. After 6 months, remodeling of the affected sternebrae and resolution of sternebral lysis were evident radiographically. Geomyces organisms and pyogranulomatous infiltrates persisted despite clinical improvement. Treatment with itraconazole was continued for an additional 3 months. CLINICAL RELEVANCE: Infection with Geomyces organisms is typically localized to the skin and nail beds. In the dog of this report, systemic dissemination of Geomyces organisms resulted in lysis of the first 2 sternebrae. Cytologic examination of fine-needle aspirates and microbial culture of samples of the affected sternebrae were important diagnostic tests for successful identification of the organism. Despite 6 months of itraconazole administration and evidence of clinical improvement, fungal organisms persisted in the dog's affected sternebrae. Practitioners should include Geomyces infection among the differential diagnoses for suspected systemic mycosis and should perform cytologic examination and microbial culture of affected tissue throughout treatment of affected dogs.

Cytologic diagnosis of diseases in reptiles.

The cytologic evaluation of samples obtained from reptile patients may provide invaluable diagnostic information to the clinician. The following article is directed toward providing information regarding the techniques used to obtain samples, discussion of sample types, and guidelines for the cytologic classification of the materials collected from tissue lesions and body fluids.

Validation of immune complex dissociation methods for use with heartworm antigen tests.

BACKGROUND: Antigen testing is routinely used to diagnose canine Dirofilaria immitis infections. Immune complex dissociation (ICD) methods, which were employed in the original heartworm antigen tests to release antigen that was bound by endogenous canine antibodies, were discontinued with improvements in assay reagents. The purpose of this study was to evaluate different ICD methods for detection of heartworm antigen by microtiter plate ELISA and assess the performance in samples from pet dogs. METHODS: The original PetChek® Heartworm Test (IDEXX Laboratories, Inc.) utilized pepsin at an acidic pH for ICD prior to antigen testing. Performance and characteristics of the pepsin ICD method were compared with those for heat treatment (with and without EDTA) and acid treatment. RESULTS: All four methods released complexed antigen in serum samples when tested using microtiter plate ELISA. Heat treatment required ≥600 µL of serum or plasma, whereas pepsin and acid methods needed only a 50-µL sample. Samples from 1115 dogs submitted to IDEXX Laboratories between 2014 and 2016 for investigation of discrepant heartworm results were evaluated with and without pepsin ICD using the PetChek Heartworm Test. Samples from 10% (n = 112) of the dogs were antigen positive with the ICD protocol only while 90% of the results remained unchanged. In a prospective study, antigen levels with and without ICD were evaluated for 12 dogs receiving pre-adulticide heartworm treatment with a macrocyclic lactone and doxycycline for 28 days. Serial samples revealed that three dogs had a reduction in detectable heartworm antigen within 4 weeks of initiating treatment. In these cases, heartworm antigen levels could be recovered with ICD. CONCLUSIONS: Heartworm antigen testing with ICD can be a valuable diagnostic tool for patients with discrepant results that have had intermittent use of a preventive, or have been treated with a macrocyclic lactone and doxycycline. Heartworm therapies may reduce antigen production and favor immune complexing in some dogs, resulting in false-negative results. Therefore, it is important to confirm positive heartworm antigen test results before initiating therapy.

Development and evaluation of a PCR assay for the detection of Cytauxzoon felis DNA in feline blood samples.

Cytauxzoonosis is an emerging tick borne infectious disease of domestic cats in the United States, caused by the organism Cytauxzoon felis (C. felis). In naturally infected domestic cats the disease is almost always fatal. Currently there are no commercially available molecular or serologic tests to facilitate the antemortem diagnosis of C. felis infection. Clinical and pathological diagnosis of cytauxzoonosis is based on microscopic identification of parasites in tissues or on blood smears. We have developed and evaluated the sensitivity and specificity of a polymerase chain reaction (PCR) based assay for the diagnosis of C. felis infections in feline blood samples. The assay is sensitive enough to detect one copy of a cloned fragment of the C. felis 18S rRNA gene. This PCR assay can be used for the rapid clinical diagnosis of cytauxzoonosis and for epidemiological studies that will better define the geographic distribution of C. felis infection in cats.

Cloning and expression of the gene encoding the major surface protein 5 (MSP5) of Anaplasma phagocytophilum and potential application for serodiagnosis.

BACKGROUND: Anaplasma phagocytophilum (formerly known as the human granulocytic ehrlichia, Ehrlichia equi and Ehrlichia phagocytophila) is an obligate intracellular organism causing clinical disease in humans and various species of domestic animals. OBJECTIVES: The objectives of this investigation were to sequence and clone the major surface protein 5 (MSP5) of A phagocytophilum and to evaluate the suitability of this antigen in the serologic diagnosis of anaplasmosis in humans and dogs. METHODS: The msp5 gene of A phagocytophilum was sequenced, cloned, and expressed in Escherichia coli. The predicted amino acid sequence homology of the various MSP5/major antigenic protein 2 orthologs was compared among various Anaplasma and Ehrlichia species. Recombinant MSP5 of A phagocytophilum was used in an ELISA to detect antibodies in serum samples from humans and dogs infected with the organism. RESULTS: Serum samples from 104 individuals previously diagnosed with A phagocytophilum infection, as well as samples from clinically healthy humans, were tested. In addition, multiple samples from 4 dogs experimentally infected with 2 different geographic isolates of A phagocytophilum and 5 dogs naturally infected with a Swiss isolate were tested using ELISA. Using this group of immunofluorescent antibody test-positive and immunofluorescent antibody test-negative samples, we found the overall agreement between assays to be >90%. CONCLUSIONS: These results indicate that recombinant MSP5 has potential for use as a diagnostic test antigen to detect infection with A phagocytophilum in both dogs and humans. However, sequence similarities among orthologs of MSP5 in related species of anaplasma and ehrlichia suggest that cross-reactivity among these pathogens is likely if the entire peptide is used as a test antigen.

Blood values in free-ranging nesting leatherback sea turtles (Dermochelys coriacea) on the coast of the Republic of Gabon.

Leatherback sea turtles (Dermochelys coriacea) are the most endangered of the seven species of sea turtles. The health status of leatherbacks is largely unknown, although the number of nesting females recorded throughout the world has decreased precipitously in the last few decades. Central African beaches may provide one of the last strongholds for nesting leatherback females. In the region, oil extraction and incidental capture pose significant threats to the health of the population. Physical examinations, hematology, plasma biochemistry, plasma corticosterone concentration, plasma protein electrophoresis, plasma vitamin concentrations, and toxicological parameters were evaluated in nesting female leatherbacks in the Republic of Gabon. The general clinical condition of the 35 turtles examined in this study was rated as good. The blood value results for a subset of these turtles are presented and compared to published results from other sea turtles. To the authors' knowledge, these are the first published baseline hematology, plasma biochemistry, and plasma protein electrophoresis values from clinically healthy nesting leatherback turtles.

Identification of functional promoters in the msp2 expression loci of Anaplasma marginale and Anaplasma phagocytophilum.

Organisms in the family Anaplasmataceae are important tick-borne pathogens of livestock worldwide and cause recently emergent infections in humans. Despite their medical importance, very little is known about how these organisms regulate gene expression in the mammalian host, the tick vector, or during transition between the host and vector. However, it is clear that gene regulation, in addition to recombinatorial mechanisms, is essential for these small genome pathogens to adapt to distinctly different environments. In this study, we identify and establish the function of three promoter elements in the locus encoding major outer membrane protein expression sites in both Anaplasma marginale and Anaplasma phagocytophilum. Gene expression from this locus involves both classical and atypical polycistronic transcripts. The identified promoter elements have a structure similar to that defined in Escherichia coli and are functional in driving protein expression in a prokaryotic cell-free transcription and translation system and in recombinant E. coli. The two strongest promoters identified in vitro and with recombinant E. coli were also shown to be functional in A. marginale infected cells, as determined by quantification of downstream transcripts. The promoters in both A. marginale and A. phagocytophilum have similar structure and activity, supporting the conclusion that the two loci are syntenic with conservation of function. In addition, they share structural elements within the promoters that appear to be likely sites for regulation. These data enhance our understanding of how expression of these variable outer membrane proteins may be controlled in the key stages of tick-borne transmission and infection.

Clinical pathology in veterinary geriatrics.

Alterations in an individual analyte rarely provide an indication of the initiating circumstances that caused the abnormality. It is obvious from the previous discussion that multiple organs or organ systems can cause abnormal results in the same analyte. This fact underscores the importance of evaluating a biochemical profile in an integrated fashion, relating abnormalities of a particular analyte with the rest of the profile as well as with the signalment, history, and physical findings in the patient. Furthermore, assessment of abnormalities should be approached with some degree of skepticism because they may not be indicative of an actual disease.

A "down under" lesion on the muzzle of a dog.

A 10-year-old, castrated, male Labrador Retriever was presented to a local veterinary practice for investigation of a firm, deeply pigmented, alopecic, subcutaneous mass (8 mm in diameter) on the left side of the muzzle. A fine-needle aspirate of the mass was submitted for cytologic evaluation to the University of Florida. Microscopically, the preparation contained a predominant population of histiocytes that contained variable numbers of intracytoplasmic, negative-staining, filamentous structures consistent with Mycobacterium sp. A presumptive diagnosis of canine leproid granuloma syndrome was based on the cytologic findings and location of the lesion. Acid-fast staining revealed bright pink, acid-fast organisms within the histiocytic cells, supporting the diagnosis. The bacteria were not detected in histopathologic sections or by a polymerase chain reaction (PCR) test 1 week later, however, possibly because of spontaneous remission. Canine leproid granuloma syndrome is a common disease in Australia, but is uncommon in dogs in North America. It is caused by a novel, unnamed Mycobacterium species and usually affects the skin and subcutaneous tissues of the head and ears. A diagnosis usually can be made in Wright's-Giemsa and acid-fast-stained cytologic specimens; however, definitive diagnosis requires PCR testing at a specialized laboratory.

Conjunctival swab cytology from a guinea pig: it's elementary!

Three 3-month-old guinea pigs (Cavia porcellus) were evaluated for purulent ocular discharge. Conjunctival swabs were obtained for cytologic evaluation of Wright's-Giemsa-stained preparations. The specimen from the most severely affected guinea pig consisted primarily of karyolytic neutrophils and small lymphocytes. Epithelial cells occasionally were observed that contained intracytoplasmic coccoid basophilic organisms, 0.5-1.5 microm in diameter. The intraepithelial inclusions were most consistent with Chlamydia sp elementary and reticulate bodies. Specimens from the other 2 guinea pigs had a similar inflammatory response, but organisms were not observed. Polymerase chain reaction (PCR) analysis of a conjunctival swab from the most severely affected guinea pig was positive for C psittaci, which also is referred to as Chlamydophila caviae, immunotype 8, formerly known as the guinea pig inclusion conjunctivitis strain of C psittaci. Chlamydial conjunctivitis is a common problem in guinea pig populations, with C caviae being specific for this species. Cytologic identification of elementary or reticulate bodies within epithelial cells is diagnostic for the organism in Giemsa-stained preparations. However, PCR is an important complementary tool when organisms are not observed and for accurate classification of the Chlamydia species.

Comparison of three enzyme-linked immunosorbant assays with the indirect immunofluorescent antibody test for the diagnosis of canine infection with Ehrlichia canis.

The aim of this study was to compare three different enzyme-linked immunosorbant assays (recombinant major antigenic protein 2 (rMAP2)-ELISA, the Immunocomb (Biogal, Israel) and the Snap 3Dx assay (IDEXX Laboratories Inc., USA)) with the indirect immunofluorescent antibody test in detecting anti-Ehrlichia canis immunoglobulin-G (IgG) antibodies. Samples tested were collected from dogs suspected to be naturally infected with E. canis and from experimentally infected dogs. When qualitative results (positive/negative) were compared, there was an overall agreement of 81% (54/67) between the indirect immunofluorescence antibody (IFA) test and the rMAP2-ELISA. An overall agreement of 94% (63/67) was found between the IFA test and the Immunocomb, and an overall agreement of 91% (61/67) was found between the IFA test and the Snap 3Dx assay. In 50 of 67 (74.6%) samples tested, complete agreement in the qualitative results was found in all four tests. Sixteen of 17 samples with disagreement in the qualitative results were found to have IFA titers of 1:320 or less. The sensitivities and specificities of the tests were found to be 0.71 and 0.85 for the rMAP2-ELISA, 0.86 and 0.98 for the Immunocomb, and 0.71 and 1.00 for the Snap 3Dx assay. The tests performed in this study were found to be highly specific in detecting E. canis antibodies. Their sensitivity was found to be low with sera having IFA titers of < or =1:320, while high with sera having titers greater than 1:320. Repeating the serological tests 1-2 weeks after the first antibody assay may overcome the sensitivity problem with titers of < or =1:320.

Differentiation of Haemobartonella canis and Mycoplasma haemofelis on the basis of comparative analysis of gene sequences.

OBJECTIVE: To determine whether Haemobartonella canis and Mycoplasma haemofelis (formerly known as H felis [large form]) can be differentiated by use of comparative analysis of gene sequences. SAMPLE POPULATION: Blood samples obtained from 3 dogs infected with H canis and 2 cats infected with M haemofelis. PROCEDURE: The partial 16S rDNA and ribonuclease P RNA (RNase P) genes were amplified, cloned, and sequenced in blood samples obtained from H canis-infected dogs and M haemofelis-infected cats. The DNA sequences were subjected to comparative analysis. RESULTS: The 16S rDNA sequences of H canis and M haemofelis were nearly identical (homology of 99.3 to 99.7%). In contrast, RNase P gene sequences had a lower degree of sequence homology between the 2 organisms (94.3 to 95.5%). CONCLUSIONS AND CLINICAL RELEVANCE: Haemobartonella canis and M haemofelis are not identical organisms. Molecular differentiation of H canis and M haemofelis is more clearly evident by use of comparative analysis of RNase P gene sequences than by comparative analysis of 16S rDNA gene sequences.