A quantitative TaqMan PCR assay for the detection of Mycoplasma suis.

AIM: To develop a TaqMan probe-based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. METHODS AND RESULTS: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay's specificity, detection limit, intra- and inter-assay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100-fold more sensitive than the cPCR. No cross-reactivity with nontarget pig mycoplasmas was observed. An average of 1·62 × 10(11) and 2·75 × 10(8) target copies ml(-1) of blood were detected in the acutely and chronically infected pigs, respectively. Three (7·5%) pigs and 32 (80·0%) sows were positive while all peccaries were negative for Myc. suis. CONCLUSION: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. SIGNIFICANCE AND IMPACT OF THE STUDY: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.

Exploratory study of Mycoplasma suis (Eperythrozoon suis) on four commercial pig farms in southern Brazil.

Mycoplasma suis (Eperythrozoon suis) was detected by PCR and Southern blot in 186 pigs (121 sows, 61 piglets and four boars) on four farms in southern Brazil. DNA was extracted from blood samples and a 16S rRNA gene fragment of M suis was amplified by PCR; Southern blot analysis was then performed on all the samples. Twenty-two of the sows (18.2 per cent) were positive by PCR, and 40 (33.1 per cent) were positive by Southern blot; only one piglet and one boar were positive. The packed cell volume and total plasma protein of the pigs and their PCR and Southern blot results were not significantly different on the four farms, but higher proportions of the pigs were positive by Southern blot than by PCR (P<0.05). The packed cell volume and total plasma protein concentrations of the M suis positive and negative sows were not significantly different.

Evaluation of an Electrochemical Point-of-Care Meter for Measuring Glucose Concentration in Blood from Periparturient Dairy Cattle.

BACKGROUND: The Precision Xtra(®) meter is a promising low cost electrochemical point-of-care unit for measuring blood glucose concentration ([gluc]) in cattle blood. The meter uses an algorithm that assumes the intra-erythrocyte [gluc] equals the plasma [gluc] on a molal basis, and that the hematocrit is similar in humans and cattle. OBJECTIVES: The primary objective was to determine the accuracy of the meter for measuring plasma [gluc] in dairy cattle. Secondary objectives were to characterize the influence of hematocrit and sample temperature on the measured value for [gluc]. ANIMALS: A total of 106 periparturient Holstein-Friesian cattle. METHODS: Blood and plasma samples (1,109) were obtained and Deming regression and Bland-Altman plots were used to determine the accuracy of the meter against the reference method (plasma hexokinase assay). Multivariable regression and linear regression were used to determine the effect of hematocrit and sample temperature on the plasma [gluc] measured by the meter. RESULTS: Intra-erythrocyte [gluc] was 18% of plasma [gluc] on a molar basis. Sample temperature had a significant linear effect on plasma [gluc] as measured by the meter for 3/5 plasma samples when measured [gluc] > 160 mg/dL. CONCLUSIONS AND CLINICAL IMPORTANCE: The meter utilizes an algorithm that is optimized for human blood and is inaccurate when applied to bovine blood. Until a cattle-specific algorithm is developed, we recommend using plasma as the analyte instead of blood and calculating plasma [gluc] using the equation: [gluc] = 0.66 × [gluc]p-meter + 15, where [gluc]p-meter is the value reported by the meter. If blood is measured, then we recommend using the equation: [gluc] = 0.90 × [gluc]b-meter + 15.

Suppression of Ia antigen expression on gamma interferon treated macrophages infected with Ehrlichia risticii.

Ehrlichia risticii is an obligate intracellular bacterium of monocytes/macrophages. In this report, using immunofluorescence staining, flow cytometry, and Kolmogorov-Smirnov analysis of histograms, the response of P338D1 and peritoneal macrophages stimulated with recombinant murine interferon-gamma (rIFN-gamma) was examined for the expression of major histocompatibility complex Class II gene product (Ia) and effect of E. risticii infection on induction of Ia surface expression. Maximal expression of Ia by sham-infected P388D1 cells was observed 2 days post rIFN-gamma addition followed by a progressive decline. These stimulatory effects of rIFN-gamma were dose dependent. Relative to sham-infected P388D1 cells, the induction of Ia by rIFN-gamma (200 U ml-1) on E. risticii-infected P388D1 cells was significantly suppressed at each time point tested through Day 5 with maximal suppression of 88% occurring on Day 2. Similarly, the induction of Ia by rIFN-gamma on E. risticii-infected peritoneal macrophages was significantly suppressed by 77% (fluorescent microscopy) when compared to sham-infected peritoneal macrophages. The higher dose of rIFN-gamma (2000 U ml-1) failed to restore Ia surface expression by E. risticii-infected P388D1 cells. The suppression of Ia on P388D1 cells in response to RIFN-gamma was not related to the degree of infection of these cells by E. risticii. A soluble inhibitor substance was not demonstrable in the supernatant from E. risticii-infected cells, nor were inhibitor levels of prostaglandin E2 levels found in the supernatant. Suppression of surface Ia expression on the macrophage suggests a mechanism whereby I. risticii may evade T-lymphocyte recognition, hinder antigen-specific T-lymphocyte activation, and promote their own survival.

Development and evaluation of a polymerase chain reaction assay using the 16S rRNA gene for detection of Eperythrozoon suis infection.

The 16S ribosomal RNA (rRNA) gene of Eperythrozoon suis was amplified using gene-specific primers developed from GenBank sequence accession U88565. The gene was subsequently cloned and sequenced. Based on these sequence data, 3 sets of E. suis-specific primers were designed. These primers selectively amplified 1394, 690, and 839 base-pair (bp) fragments of the 16S rRNA gene from DNA of E. suis extracted from the blood of an experimentally infected pig during a parasitemic episode. No polymerase chain reaction (PCR) products were amplified from purified DNA of Haemobartonella felis, Mycoplasma genitalium, or Bartonella bacilliformis using 2 of these primer sets. When the primer set amplifying the 690-bp fragment was used, faint bands were observed with H. felis as the target DNA. No PCR products were amplified from DNA that had been extracted from the blood of a noninfected pig or using PCR reagents without target DNA. The detection limits for E. suis by competitive quantitative PCR were estimated to range from 57 and 800 organisms/assay. This is the first report of the utility of PCR-facilitated diagnosis and quantitation of E. suis based on the 16S rRNA gene. The PCR method developed will be useful in monitoring the progression and significance of E. suis in the disease process in the pig.

Detection of Haemobartonella felis in cats with experimentally induced acute and chronic infections, using a polymerase chain reaction assay.

OBJECTIVE: To develop a test for detection of Haemobartonella felis, using a polymerase chain reaction (PCR) assay. ANIMALS: 4 adult cats seronegative for FeLV and feline immunodeficiency virus. PROCEDURE: Cats were infected with H felis by i.v. administration of 1 ml of blood obtained from an infected cat. Rectal temperature, PCV, and microscopic examination of blood smears for organisms were monitored daily. At peak of infection, doxycycline treatment was initiated for 21 days. Blood samples were collected at weekly intervals. Six months after treatment, 2 cats were given methylprednisolone (14 mg/kg of body weight, i.m.). Daily blood samples were collected for CBC, detection of organisms, and PCR evaluation. On the basis of the 16S rRNA gene sequence of H felis, specific PCR primers were created for a 393-basepair internal fragment. RESULTS: The 393-basepair product was consistently amplified from blood samples obtained during peak parasitemia but not during the last week of or immediately after completion of doxycycline treatment. After treatment, PCV returned to the reference range, and organisms were not observed in blood samples; however, the PCR product could be consistently amplified. After administration of methylprednisolone, organisms were only rarely observed in blood smears but were consistently detected by PCR analysis. CLINICAL RELEVANCE: Using PCR analysis, it was possible to detect H felis in blood samples obtained from cats during peak parasitemia, during most of the carrier phase, and after challenge with immunosuppressive drugs. During and immediately after antibiotic treatment, this test may fail to detect the organisms.

Use of a polymerase chain reaction assay for detection of Haemobartonella canis in a dog.

A polymerase chain reaction (PCR) originally developed for detection of Haemobartonella felis in cats was successfully used for detection of H canis in an 8-year-old spayed Great Dane. The dog had been splenectomized and was undergoing immunosuppressive chemotherapy at the time of diagnosis. Sequence analysis of the 16S ribosomal RNA gene revealed that the Haemobartonella spp infecting this dog was 97% homologous to the sequence previously reported for the Ohio strain of H felis. Clinical and hematologic abnormalities as well as identification of the organisms by use of light and electron microscopy supported the diagnosis of H canis. The PCR assay used for detection of H felis may be useful for the detection of H canis in dogs prior to blood donation, splenectomy, or treatment with immunosuppressive drugs.

Clostridium botulinum type B toxicosis in a herd of cattle and a group of mules.

Clostridium botulinum type B toxicosis was diagnosed as the cause of generalized weakness and death in a group of cows and mules fed from the same batch of rye silage. One severely affected cow was treated and recovered, as did other less severely affected cows. All affected mules died. The remaining cattle in the herd were then vaccinated before continued feeding of the silage.

Leukopenia in six healthy Belgian Tervuren.

A healthy 6.5-year-old sexually intact female Belgian Tervuren was found to be persistently leukopenic during preoperative evaluation for routine ovariohysterectomy. Abnormalities of the erythroid or myeloid series were not detected during bone marrow analysis. Blood samples for CBC were collected from 8 additional healthy Belgian Tervuren of both sexes and of various ages. Six of the 9 dogs were leukopenic, with WBC counts between 2.38 and 5.42 x 10(3) WBC/microl (mean +/- SD, 4.13 +/- 1.04 x 10(3) WBC/microl). Leukopenia was a persistent finding in the 3 dogs from which multiple blood samples were collected. All dogs were otherwise clinically normal. Leukopenia, as defined by a WBC count < 6.00 x 10(3) WBC/microl, may be a common finding in the Belgian Tervuren breed.

Results of hematologic analyses and prevalence of physiologic leukopenia in Belgian Tervuren.

OBJECTIVE: To determine reference ranges for results of hematologic analyses of healthy Belgian Tervuren, to compare results of hematologic analyses for healthy Belgian Tervuren with results for healthy dogs of other breeds, and to determine prevalence of physiologic leukopenia in Belgian Tervuren. DESIGN: Cohort study. ANIMALS: 180 healthy Belgian Tervuren and 63 healthy dogs of other breeds. PROCEDURE: Blood samples were analyzed by use of an automated device. Reference ranges were calculated for Belgian Tervuren by use of standard methods. RESULTS: Total WBC counts of Belgian Tervuren ranged from 2.61 to 16.90 x 10(3)/microl. Mean WBC count of Belgian Tervuren (mean +/- SEM, 7.04 +/- 0.16 x 10(3)/microl) was significantly lower than mean count for control dogs. Significantly more Belgian Tervuren (65/180; 36%) than control dogs (2/63; 3%) had WBC counts < 6.00 x 10(3)/microl. Percentage of Belgian Tervuren with WBC count < 6.00 x 103/microl was low for dogs < or = 2 years old, increased sharply for dogs between 2 and 4 years old, and was approximately 65% for dogs > 4 years old. Neutrophil, lymphocyte, and monocyte counts were significantly lower, and RBC count, hematocrit, and eosinophil fraction were significantly higher in Belgian Tervuren than in control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that physiologic leukopenia, resulting from low numbers of neutrophils, lymphocytes, and monocytes, may be a typical finding in a large percentage of healthy Belgian Tervuren and is not of clinical importance in otherwise healthy dogs. Healthy Belgian Tervuren may also have RBC counts and hematocrits higher than expected for healthy dogs.

Light and electron microscopic features of eperythrozoon-like parasites in a North American opossum (Didelphis virginiana).

Epierythrocytic parasites associated with a severe anemic episode have not been previously reported in the opossum. A Wright-Giemsa-stained peripheral blood smear from an anemic North American opossum (Didelphis virginiana) revealed numerous organisms attached to red blood cells either singularly or in chains. Ring forms of the organism were common and could be found free in the plasma. Electron microscopy revealed that these organisms were attached to the intact plasma membrane in depressions on the surface of red blood cells. Delicate fibrils between the organism and adjacent membrane were observed. The organisms were round to oval with a diameter of 300-750 nm and were enclosed by a single limiting membrane. The light and electron microscopic features of these epierythrocytic organisms are similar to those reported for Eperythrozoon and Haemobartonella species.

Detection of Bartonella spp. in neotropical felids and evaluation of risk factors and hematological abnormalities associated with infection.

Although antibodies to Bartonella henselae have been described in all neotropical felid species, DNA has been detected in only one species, Leopardus wiedii. The aim of this study was to determine whether DNA of Bartonella spp. could be detected in blood of other captive neotropical felids and evaluate risk factors and hematological findings associated with infection. Blood samples were collected from 57 small felids, including 1 Leopardus geoffroyi, 17 L. wiedii, 22 Leopardus tigrinus, 14 Leopardus pardalis, and 3 Puma yagouaroundi; 10 blood samples from Panthera onca were retrieved from blood banks. Complete blood counts were performed on blood samples from small felids, while all samples were evaluated by PCR. DNA extraction was confirmed by amplification of the cat GAPDH gene. Bartonella spp. were assessed by amplifying a fragment of their 16S-23S rRNA intergenic spacer region; PCR products were purified and sequenced. For the small neotropical felids, risk factors [origin (wild-caught or zoo-born), gender, felid species, and flea exposure] were evaluated using exact multiple logistic regression. Hematological findings (anemia, polycythemia/hyperproteinemia, leukocytosis and leukopenia) were tested for association with infection using Fisher's exact test. The 635bp product amplified from 10 samples (10/67=14.92%) was identified as B. henselae by sequencing. Small neotropical felid males were more likely to be positive than females (95% CI=0.00-0.451, p=0.0028), however other analyzed variables were not considered risk factors (p>0.05). Hematological abnormalities were not associated with infection (p>0.05). This is the first report documenting B. henselae detection by PCR in several species of neotropical felids.

Characterization of Ehrlichia risticii binding, internalization, and proliferation in host cells by flow cytometry.

The binding, internalization, and proliferation of Ehrlichia risticii in P388D1 cells and equine polymorphonuclear (PMN) leukocytes were studied by immunofluorescent staining and flow cytometric analysis. The binding of ehrlichiae to P388D1 cells at 4 degrees C was dose dependent, and the antigens of bound organisms were susceptible to pronase treatment. Additionally, the binding of ehrlichiae to P388D1 cells was diminished when either P388D1 cells or ehrlichiae were treated with 1% paraformaldehyde for 30 min or 0.25% trypsin for 15 min. These results indicate that the ehrlichial ligand and host cell receptor are likely surface proteins. Following incubation at 37 degrees C, bound E. risticii and/or its antigens were removed with pronase and indirect immunofluorescent staining in the presence of saponin was used to examine intracellular ehrlichiae. Our results indicate that E. risticii was internalized into P388D1 cells within 3 h and proliferated by 48 h of incubation. The microfilament-disrupting agent cytochalasin D and the transglutaminase inhibitor monodansylcadaverine were used to differentiate between phagocytosis (sensitive to cytochalasin) and receptor-mediated endocytosis (sensitive to monodansylcadaverine) of E. risticii by P388D1 cells. In concentrations that produced distinctive morphological changes and inhibited phagocytosis of polystyrene latex beads, cytochalasin D did not suppress the infectivity of E. risticii. Binding, internalization, or proliferation of E. risticii was not affected by cytochalasin D. However, monodansylcadaverine inhibited infection of E. risticii in a dose-dependent manner. The agent did not affect the attachment of ehrlichiae to host cells, but it did suppress internalization and proliferation. These results suggest that E. risticii is internalized by receptor-mediated endocytosis and that productive infection by E. risticii does not depend on phagocytosis by the P388D1 cells. Although E. risticii did not bind to the surface of equine PMN leukocytes at 4 degrees C, organisms were taken up by this cell at 37 degrees C. E. risticii, however, failed to survive in equine PMN leukocytes.

Inhibition of binding, entry, or intracellular proliferation of Ehrlichia risticii in P388D1 cells by anti-E. risticii serum, immunoglobulin G, or Fab fragment.

The effects of equine antiserum, immunoglobulin G (IgG) specific for Ehrlichia risticii, and its Fab fragment on E. risticii binding to, internalization into, and proliferation in P388D1 cells were studied by immunofluorescence flow cytometry. Anti-E. risticii equine serum or IgG inhibited E. risticii at a stage beyond binding and internalization. In contrast, monovalent anti-E. risticii equine Fab fragments inhibited E. risticii binding and internalization into P388D1 cells. In the presence of control equine serum, IgG, or its Fab fragment, E. risticii cells were bound, were internalized and subsequently grew within P388D1 cells, and eventually destroyed the host cells as effectively as was the case without equine serum, IgG, or Fab fragments. Anti-E. risticii IgG but not normal horse IgG inhibited L-[14C]glutamine metabolism in Percoll gradient-purified E. risticii. These findings suggest that the Fab fragment of intact anti-E. risticii IgG blocks the ligands on E. risticii responsible for non-IgG-mediated internalization and diverts them to bind via the Fc receptor. Following Fc-mediated entry of E. risticii, the antibody interfered with the metabolic activity of E. risticii cells, rendering them incapable of proliferation in P388D1 cells and resulting in the eventual destruction of the organisms.

Presence of parasite antigen on the surface of P388D1 cells infected with Ehrlichia risticii.

Indirect immunofluorescence staining of macrophages infected with Ehrlichia risticii by anti-E. risticii serum revealed a punctate staining pattern on the surface of the host cell. This pattern was distinguishable by fluorescence microscopy from E. risticii bound to the surface of the macrophage and from intracellular E. risticii. The surface localization of ehrlichial antigen on infected macrophages was confirmed by electron microscopy with immunoferritin labeling. As the intracellular ehrlichial burden increased, the amount of ehrlichial antigen on the host cell surface increased. Prokaryotic protein synthesis was necessary for the maintenance of ehrlichial antigen on the host cell surface, as demonstrated by disappearance of the surface antigen following treatment with oxytetracycline. However, host cell protein synthesis was not required, as demonstrated by the continued presence of ehrlichial antigen on the surface of host cells after cycloheximide treatment. Pronase treatment abolished the ehrlichial antigen present on the cell surface, indicating that this antigen is a protein. Anti-E. risticii serum or immunoglobulin G-mediated antibody-dependent cellular cytotoxicity of infected cells was demonstrated in a chromium release assay. These results imply that the parasite antigen on the host cell surface has a role in the pathogenesis of ehrlichiosis.

Development and evaluation of a PCR-based assay for detection of Haemobartonella felis in cats and differentiation of H. felis from related bacteria by restriction fragment length polymorphism analysis.

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats.

Genome size of Eperythrozoon suis and hybridization with 16S rRNA gene.

The genome size of Eperythrozoon suis, an unculturable haemotropic mycoplasma, was estimated using pulsed-field gel electrophoresis (PFGE). Gamma irradiation was used to introduce one (on the average) double-strand break in the E. suis Illinois chromosome. Restriction enzymes that cut infrequently were also used to analyze genome size. The size estimate for the full-length genome was 745 kilobases (kb), whereas the size estimates based on the summation of restriction fragments ranged from 730 to 770 kb. The 16S rRNA gene was located on the 120-kb MluI fragment, 128-kb NruI fragment, 25-kb SacII fragment, and 217-kb SalI fragment by Southern blotting.

Specific in situ hybridization of Haemobartonella felis with a DNA probe and tyramide signal amplification.

Haemobartonella felis is an epierythrocytic bacterium suspected to be the causative agent of feline infectious anemia. Previous studies with a polymerase chain reaction assay have identified a mycoplasmal 16S rRNA gene sequence that coincides with clinical disease and the presence of organisms in the blood. Tissues from a cat experimentally infected with H. felis were used for in situ hybridization studies to physically link this 16S rRNA gene to the organisms on the red cells. A biotin-labeled probe was used in conjunction with tyramide signal amplification to visualize the hybridization signal. This study clearly demonstrates a specific hybridization signal on the red cells in the tissues of the H. felis-infected cat. This in situ hybridization study is the final step in fulfilling the molecular guidelines for disease causation and proves that H. felis, a mycoplasmal organism, is the causative agent of feline infectious anemia.

Necrotizing mycotic vasculitis with cerebral infarction caused by Aspergillus niger in a horse with acute typholocolitis.

An 18-year-old Morgan mare was presented to the Veterinary Medical Teaching Hospital, University of Illinois, with a 10-day history of watery diarrhea, depression, and dysphagia. On admission, the animal was severely dehydrated, depressed, and unable to swallow and had no clinical signs of diarrhea. The respiratory and heart rate and body temperature were within normal limits. Following fluid therapy, the mare developed severe watery diarrhea and continued to be depressed, incoordinated, and dysphagic. The animal died on the fourth day after admission and was sent to the Laboratories of Veterinary Diagnostic Medicine for necropsy. Gross postmortem findings were consistent with an acute cerebral infarction in the right cerebral hemisphere, an acute necrotizing typhlocolitis, multifocal petechial and ecchymotic hemorrhages, enlarged and congested pars intermedia of the pituitary gland, and marked bilateral adrenocortical hyperplasia with multifocal areas of necrosis and hemorrhage. Histologic evaluation of the affected brain demonstrated an area of coagulative necrosis of the gray matter, with hemorrhage, vasculitis, and thrombosis. There were many fungal hyphae 3.5-6.0 microm, pale basophilic, septate, and occasionally branching at 45 degrees present in the arterial walls and throughout the necrotic tissue. Immunohistochemical analysis revealed Aspergillus niger as the etiologic agent responsible for the mycotic vasculitis and infarction in the brain. Bacteria culture and immunohistochemical staining of the colon and cecum failed to demonstrate specific pathogens.

Can we continue research in splenectomized dogs? Mycoplasma haemocanis: old problem--new insight.

We report the appearance of a Mycoplasma haemocanis infection in laboratory dogs, which has been reported previously, yet, never before in Europe. Outbreak of the disease was triggered by a splenectomy intended to prepare the dogs for a hemorrhagic shock study. The clinical course of the dogs was dramatic including anorexia and hemolytic anemia. Treatment included allogeneic transfusion, prednisone, and oxytetracycline. Systematic follow-up (n = 12, blood smears, antibody testing and specific polymerase chain reaction) gives clear evidence that persistent eradication of M. haemocanis is unlikely. We, therefore, had to abandon the intended shock study. In the absence of effective surveillance and screening for M. haemocanis, the question arises whether it is prudent to continue shock research in splenectomized dogs.