Spatial distribution of seroprevalence for Anaplasma phagocytophilum, Borrelia burgdorferi, Ehrlichia canis, and Dirofilaria immitis in dogs in Washington, Oregon, and California

Background: In the US little spatially defined information regarding exposure to most vector‐borne pathogens in dogs is available for the states of California (CA), Oregon (OR), and Washington (WA). Objectives: The purpose of the present study was to evaluate the spatial distribution of seroprevalence for 4 vector‐borne pathogens, Anaplasma phagocytophilum, Borrelia burgdorferi, Ehrlichia canis, and Dirofilaria immitis, across the 3 western coastal states of the contiguous United States that extend from the northern Mexican to the southern Canadian border. Methods: A convenience sample, targeting blood from 20 pet dogs per county across CA, OR, and WA, was evaluated using a canine point‐of‐care ELISA kit. Geographic coordinates of home zip code were displayed using a geographic information system. A total of 2431 dogs from CA, OR, and WA were tested. Results: The overall seroprevalence was highest for A. phagocytophilum (2.4%), followed by B. burgdorferi (1.2%), and E. canis (0.7%). The prevalence of infection with D. immitis was 0.7%. At the individual dog level, there was a significant association between seropositivity to B. burgdorferi and A. phagocytophilum (odds ratio=18.7, 95% confidence interval=6.8–47.1). For most positive results, prevalence tended to decrease with increasing latitude; thus, the highest rates of seropositivity occurred in CA, followed by OR, and then WA; one exception was seropositivity for B. burgdorferi, which was higher in WA (0.38%) than in OR (0.15%), but considerably lower than in CA (2.00%). In WA, dogs that tested positive for A. phagocytophilum, E. canis, and B. burgdorferi were in the southern Puget Sound area. For D. immitis, none of the dogs in WA was positive. Conclusions: Seropositivity for vector‐borne pathogens is broadly but patchily distributed in dogs in CA, OR, and WA.     

Timing of seroconversion and acquisition of positive polymerase chain reaction assay results in calves experimentally infected with bovine leukemia virus

OBJECTIVE: To determine the interval to provirus and serum antibody detection (via PCR assay and ELISA, respectively) in calves after experimental inoculation with bovine leukemia virus (BLV). ANIMALS: 8 colostrum-deprived, BLV-negative Holstein bull calves (> or = 6 weeks old). PROCEDURES: Via IM injection, each calf received a fresh whole-blood inoculum (day 0) calculated to contain 2 x 10(6) lymphocytes. Blood samples for the ELISA and PCR assay were collected from calves immediately prior to inoculation and weekly thereafter for 7 weeks. Mean and median number of weeks to PCR-detected conversion of BLV status and seroconversion were calculated. Point sensitivity and cumulative sensitivity of the 2 assays were calculated at each sample collection. At each sampling time, the proportion of calves identified as infected by the cumulative weekly ELISA and PCR assay results was compared by use of a Fisher exact test. RESULTS: In 5 calves, conversion of BLV status was detected via PCR assay before seroconversion was identified. However, seroconversion preceded PCR-detected conversion in 2 calves. In 1 calf, both assays yielded positive results at the same test date. These differences were not significant. CONCLUSIONS AND CLINICAL RELEVANCE: In experimentally inoculated BLV-negative calves, conversion of BLV status was detected via PCR assay more quickly than via ELISA; this difference was not significant and probably not clinically important. The PCR assay may be useful as a confirmatory test in animals of exceptional value; tests based on viral identification may become critically important if vaccines against BLV infection are developed and marketed.     

Evaluation and use of a nested polymerase chain reaction assay in cats experimentally infected with Bartonella henselae genotype I and Bartonella henselae genotype II.

Cats have been shown to be infected with Bartonella henselae genotype I, B. henselae genotype II, and B. clarridgeiae. Feline bartonellosis infections and the strains involved in these infections are important in both veterinary and human medicine. Nucleic acid amplification methods such as polymerase chain reaction (PCR) are being used in both research and diagnostics as tools for understanding many infectious diseases. Bartonella bacteremia in cats is detected by blood culture; however, because of the limitations of culture (delayed turnaround time and sensitivity limits), PCR may be a more efficient method for identifying infected cats. Three distinct PCR assays that could differentiate among B. henselae genotype I, B. henselae genotype II. and B. clarridgeiae were developed and used to detect as few as 3.2 organisms. Fourteen cats experimentally infected with B. henselae genotype I and B. henselae genotype II were followed by bacterial culture and PCR through the course of infection, including periods of primary and relapsing bacteremia. The PCR assay was positive in 11 of the 14 cats for periods of 1-9 weeks after culture became negative. Of the 223 blood specimens that were culture negative, the PCR assay was positive in 38 (17%) of the specimens. Two of the 14 cats developed relapsing bacteremia. The 2 B. henselae genotypes were amplified in the cats and the bacteremic phase of these infections as determined by PCR lasted for a longer period than previously determined by culture. Using laboratory assays such as PCR to understand the strains involved in feline bartonellosis and the course of the infection is important in the understanding of these zoonotic agents.     

Use of the polymerase chain reaction assay for the detection of Mycobacterium avium subspecies paratuberculosis in blood and liver biopsies from experimentally infected sheep

OBJECTIVE: To evaluate a polymerase chain reaction-based assay for the detection of Mycobacterium avium subsp paratuberculosis in blood and liver biopsies from subclinically infected sheep. STUDY DESIGN: A direct PCR assay for the detection of M a paratuberculosis was applied to liver biopsy specimens and to samples of blood that were sequentially collected over 53 weeks from 14 sheep infected experimentally with the organism. RESULTS: Of 117 blood samples from the 14 experimentally infected sheep, two tested positive in the PCR assay. Both positive results were obtained in two subclinically infected sheep that had paratuberculosis later confirmed by histological examination at necropsy. However, the assay failed to detect the target DNA in samples of blood from five other sheep with histologically confirmed paratuberculosis. Similarly, the PCR assay on liver biopsy specimens collected 32 weeks after administration of M a paratuberculosis gave only two positive results, both of which were obtained in sheep with histological evidence of paratuberculosis. CONCLUSIONS: The PCR assay on blood and liver biopsies does not provide a useful method for the diagnosis of M a paratuberculosis infection in subclinically infected sheep.     

Study of equid herpesviruses 2 and 5 in Iceland with a type-specific polymerase chain reaction

The horse population in Iceland is a special breed, isolated from other horses for at least 1000 years. This provides an exceptional opportunity to investigate old and new pathogens in an inbred herd with few infectious diseases. We have developed a high sensitivity semi-nested PCR to study equid gammaherpesviruses 2 and 5 (EHV-2 and 5) in Iceland. The first PCR is group specific, the second type-specific, targeting a 113bp sequence in the glyB gene. DNA isolated from white blood cells and 18 different organs was tested for the presence of EHV-2 and 5. This was done in adult horses and foals, healthy and with various enteric infections. Both virus types were easily detected in all types of organs tested or EHV-2 in 79% cases and EHV-5 in 63%. In DNA from PBMC or buffy-coat EHV-2 was found in 20% cases and EHV-5 in 10%, all except one positive were foals. Co-culture of PBMC on fetal horse kidney cells was efficient for detecting EHV-2 but not for EHV-5. We verify here for the first time infections with EHV-2 and 5 in horses in Iceland and show that both viruses are common.     

Looking for prognosticators in ovine anaplasmosis: discriminant analysis of clinical and haematological parameters in lambs belonging to differently susceptible breeds experimentally infected with Anaplasma ovis

Background

A study was carried out to evaluate the response of different native sheep breeds to experimental infection with Anaplasma ovis, the most prevalent sheep tick-borne pathogen in Apulia (Southern Italy). Thirty-four lambs belonging to a Northern European breed (Suffolk) and two Southern Italian breeds (Comisana and Altamurana) were infected. Eleven clinical as well as haematological parameters were monitored at different temporal resolutions on the same subjects before and after the infection, resulting in a data set of 435 observations. The present work, aiming to further the research, presents the results of a multivariate analysis carried out to identify which parameters out of the eleven considered are the most reliable parameters to be considered as markers of the disease phenotype as well as prognosticators of practical clinical importance.

Results

Data were analysed by discriminant analysis. Out of the eleven considered variables (red blood cells, packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin content, haemoglobin concentration, white blood cells, neutrophils, leukocytes, platelets, rectal temperature), only seven were included in the step-wise model since significantly increasing the Mahlanobis distance between the two closest groups. Both discriminant functions resulted to be highly significant (P < 0.0001) and the percentage of variation accounted for by the first discriminant function was 63.6% of the variance in the grouping variable.

Conclusions

Taken together, the observed results stress the marked differentiation among the three breeds in terms of physio-pathological phenotypes indicating packed cell volume and red blood cell count as the most informative parameters in the routine clinical practice for A. ovis infection in sheep.     

Severity and persistence of footrot in Merino sheep experimentally infected with a protease thermostable strain of Dichelobacter nodosus at five sites

Objective To test the hypothesis that ovine footrot associated with a thermostable protease strain of Dichelobacter nodosus undergoes self cure or is sustained as an annually recurring disease, depending on the environment.
Design and procedure Forty Merino sheep from a single blood line were infected with a protease thermostable strain of D nodosus a t each of five sites in Western Australia. Footrot lesions and microscopic evidence of D nodosus were recorded every fortnight for 2.5 years, supplemented by laboratory culture. Rainfall, soil and air temperature, pasture quantity and composition and soil types were also recorded. Flocks that apparently self cured were relocated to a more favourable site for footrot in the final spring season.
Results The maximum prevalence of feet with clinical footrot lesions was 80.6, 1.3, 14.4, 3.8 and 88.1% at the five sites. Severe footrot occurred for three consecutive spring seasons at one site that had clay loam soil and at least 3500 kg/ha total pasture dry matter annually. However, the infection was asymptomatic for up to 10 weeks between outbreaks. D nodosus was isolated from flocks for 2.5 years at only two sites, although there was microscopic evidence of the organism at other sites in the final year. A thermolabile variant (strain U6) of D nodosus was isolated from the two sites where footrot persisted.
Conclusion Depending on time and location, ovine footrot induced by a protease thermostable strain of D nodosus either self cured or persisted as annual outbreaks interspersed with periods of asymptomatic infection.     

Comparison of bacterial culture, polymerase chain reaction, and a mix-enzyme-linked immunosorbent assay for the detection of Salmonella status in grow-to-finish pigs in western Canada with a Bayesian approach

Among grow-to-finish pigs from 10 herds in Alberta and Saskatchewan, 23 (16%) of 144 fecal samples were culture-positive and 40 (28%) of 144 pigs were seropositive for Salmonella. With a Bayesian model specifying dependence between the 2 tests, the sensitivity (Se) of culture and real-time polymerase chain reaction (RT-PCR) was 79% to 86%, depending on the cut-off value for the enzyme-linked immunosorbent assay (ELISA). Culture specificity (Sp) was assumed to be 100%; RT-PCR Sp was found to be 94%. The ELISA Se was 76% and 51% at optical density cut-off values ≥ 20% and ≥ 40%, respectively; the Sp was 94% at each cut-off value. The model showed some sensitivity to ELISA prior information, the ELISA Se being approximately 8% lower when informative prior information was specified in the model. When there was no adjustment for dependence between culture and RT-PCR, the posterior estimates for both culture and RT-PCR Se were 11% higher than with the conditional-dependence model and had considerably narrower probability intervals, which suggests that correlation between culture and PCR is important and should be adjusted for in future studies.     

Evaluation of cytoreductive efficacy of vincristine, cyclophosphamide, and Doxorubicin in dogs with lymphoma by measuring the number of neoplastic lymphoid cells with real-time polymerase chain reaction

Background: The cytoreductive efficacy of the individual components of multidrug chemotherapy for canine lymphoma is difficult to evaluate after complete remission. Objectives: To compare the cytoreductive efficacy of vincristine (VCR), cyclophosphamide (CPA), and doxorubicin (DXR) in dogs that received a 6‐month modified version of the University of Wisconsin‐Madison chemotherapy protocol (UW‐25). Animals: Twenty‐nine dogs with high‐grade B‐cell multicentric lymphoma. Methods: Rearranged immunoglobulin heavy chain gene fragments from lymphoma cells were amplified by polymerase chain reaction (PCR) and sequenced to prepare clone‐specific primers and probes for real‐time PCR. The number of lymphoma cells in peripheral blood was measured from diagnosis to week 11 of UW‐25. Results: The number of lymphoma cells after the 1st administration of VCR, CPA, and DXR in weeks 1–4 was decreased in 29/29 (100%), 15/29 (51.7%), and 26/27 (96.3%) dogs, respectively. The cytoreductive efficacy of CPA was less than that of VCR and DXR. VCR, CPA, and DXR administered in weeks 6–9 were effective in 5/26 (19.2%), 5/20 (25.0%), and 14/19 (73.7%) dogs, respectively, indicating the sustained cytoreductive efficacy of DXR. CPA nonresponders were heavier and exhibited a shorter 1st remission than CPA responders. Conclusion and Clinical Importance: When using UW‐25 for treatment of canine lymphoma, CPA was found to have less cytoreductive efficacy than VCR and DXR. Real‐time PCR‐based quantification of tumor cells is an objective marker of the efficacy of chemotherapeutic agents.     

Identification of differentially expressed genes in ileum, intestinal lymph node and peripheral blood mononuclear cells of sheep infected with Mycobacterium avium subsp. paratuberculosis using differential display polymerase chain reaction

Discovery of differentially expressed genes aids in understanding molecular mechanisms underpinning normal and pathological states. When studying animals such as sheep where the entire genome has not been characterized, techniques that do not require knowledge of gene sequences are particularly advantageous. We used one such technique, differential display polymerase chain reaction (DD-PCR), to identify genes that had different degrees of expression in response to Mycobacterium avium subsp. paratuberculosis (M. ptb), the organism that causes Johne's disease in ruminants. Differentially expressed genes were validated by quantitative PCR using especially selected reference genes established in this study. Sheep (n = 47) were classified according to history of exposure to M. ptb and infection status by histology and faecal and tissue culture. Differences in levels of gene expression were analyzed using restricted maximum likelihood (REML) in a linear mixed model. Five genes from the ileum and 17 genes from lymph node were differentially expressed in ovine Johne's disease. Expression of seven of these genes was also significantly different in peripheral blood mononuclear cells. Genes identified in association with M. ptb infection had a wide range of functions in pathways including: antigen presentation, signal transduction and cell differentiation, TLR signaling, immune cell activation and chemokine functions, granulomatous inflammation, Th1 suppression and apoptosis.     

Successful treatment and polymerase chain reaction (PCR) confirmation of Tyzzer's disease in a foal and clinical and pathologic characteristics of 6 additional foals (1986-2005)

BACKGROUND: Tyzzer's disease is a rapidly progressive and highly fatal hepatitis of foals caused by Clostridium piliforme. Survival of a confirmed case has not been reported previously. HYPOTHESIS: Successful therapy of C. piliforme infection in foals is possible. Polymerase chain reaction (PCR) can be used to diagnose Tyzzer's disease antemortem or postmortem. ANIMALS: Seven foals were included in the study. METHODS: Retrospective study was made to evaluate the clinical and pathologic characteristics of foals with Tyzzer's disease. Medical records of the Veterinary Medical Teaching Hospital at University of California Davis were reviewed. Foals <3 months old were included in the study if typical clinical signs were present and histologic examination identified multifocal coagulative necrosis and hepatitis with intracytoplasmic filamentous bacilli, consistent with C. piliforme. A real-time TaqMan assay was developed to detect C. piliforme gene sequences in liver tissue from affected foals. RESULTS: Median survival time from onset of disease in nonsurviving foals was 30 hours (mean 34.5 +/- 20.1; range, 16-62 hours). Common clinical findings included lethargy, recumbency, seizures, and fever. Laboratory findings included metabolic acidosis, hypoglycemia and increased activity of hepatobiliary enzymes. Treatment consisted of IV fluids, antimicrobial and antiinflammatory drugs, and parenteral nutrition. One filly survived, whereas 6 died. Postmortem examination of the 6 foals that died disclosed hepatomegaly with multifocal necrosis. Liver tissue from 4 foals was positive for C. piliforme gene sequences using PCR. CONCLUSIONS AND CLINICAL IMPORTANCE: Although the mortality rate of Tyzzer's disease is high, successful outcome is possible if intensive care is initiated promptly. PCR can be used for early and specific diagnosis.     

Comparison of fecal culture and F57 real-time polymerase chain reaction for the detection of Mycobacterium avium subspecies paratuberculosis in Swiss cattle herds with a history of paratuberculosis

Background

Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP.

Results

By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%).

Conclusions

There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease.     

Evaluation of a porcine circovirus type 2-specific antigen-capture enzyme-linked immunosorbent assay for the diagnosis of postweaning multisystemic wasting syndrome in pigs: comparison with virus isolation, immunohistochemistry, and the polymerase chain reaction.

Quantitative virus isolation, immunohistochemistry, polymerase chain reaction (PCR) assay, and a porcine circovirus 2 (PCV2)-specific antigen-capture enzyme-linked immunosorbent assay (ELISA) were used for differentiation between clinical and subclinical PCV2 infections of swine. Tissue samples from pigs experimentally infected with PCV2 and field cases of postweaning multisystemic wasting syndrome and PCV2-associated reproductive disorders were used in this evaluation. In initial studies on 6 PCV2 pools using 3 previously published PCR protocols for PCV2 detection, quantitative virus isolation, and antigen-capture ELISA, substantial differences in sensitivity were identified among these procedures. Examination of tissue samples from diseased and clinically normal pigs indicated that immunohistochemistry, quantitative virus isolation, and antigen-capture ELISA could be used to differentiate between clinical and subclinical PCV2 infections, but the PCR assay could not. Because subclinical infections of pigs with PCV2 are common, the use of nonquantitative PCR as a diagnostic tool for PCV2-related diseases should be discouraged and the PCV2-specific antigen-capture ELISA evaluated further.     

Plasma drug concentrations and clinical effects of a peripheral alpha‐2‐adrenoceptor antagonist,MK‐467, in horses sedated with detomidine

ObjectiveTo investigate plasma drug concentrations and the effect of MK-467 (L-659′066) on sedation, heart rate and gut motility in horses sedated with intravenous (IV) detomidine.Study designExperimental randomized blinded crossover study.AnimalsSix healthy horses.MethodsDetomidine (10 μg kg^?1 IV) was administered alone (DET) and in combination with MK-467 (250 μg kg^?1 IV; DET + MK). The level of sedation and intestinal sounds were scored. Heart rate (HR) and central venous pressure (CVP) were measured. Blood was collected to determine plasma drug concentrations. Repeated measures anova was used for HR, CVP and intestinal sounds, and the Student's t-test for pairwise comparisons between treatments for the area under the time-sedation curve (AUC_sed) and pharmacokinetic parameters. Significance was set at p < 0.05.ResultsA significant reduction in HR was detected after DET, and HR was significantly higher after DET + MK than DET alone. No heart blocks were detected in any DET + MK treated horses. DET + MK attenuated the early increase in CVP detected after DET, but later the CVP decreased with both treatments. Detomidine-induced intestinal hypomotility was prevented by MK-467. AUC_sed was significantly higher with DET than DET + MK, but maximal sedations scores did not differ significantly between treatments. MK-467 lowered the AUC of the plasma concentration of detomidine, and increased its volume of distribution and clearance.Conclusions and clinical relevanceMK-467 prevented detomidine induced bradycardia and intestinal hypomotility. MK-467 did not affect the clinical quality of detomidine-induced sedation, but the duration of the effect was reduced, which may have been caused by the effects of MK-467 on the plasma concentration of detomidine. MK-467 may be useful clinically in the prevention of certain peripheral side effects of detomidine in horses.