Assessment of the effect of long-term serum storage for retrospective serologic diagnosis of canine monocytic ehrlichiosis (Ehrlichia canis).

There is currently sparse information on the possible effect of long-term storage of serum specimens for the retrospective serodiagnosis of canine monocytic ehrlichiosis (CME). The aim of this study was to assess the agreement between the original serologic outcome and the results of a repeat indirect fluorescent antibody (IFA) assay for the detection of IgG antibodies against E. canis. A secondary aim was to compare the diagnostic performance of two commercially available point-of-care (POC) immunochromatographic (IC) assays. Archived serum samples originally tested as positive (n=66) or negative (n=19) for E. canis IgG antibodies and kept frozen at -20°C for a median of 22 years, were retrospectively examined by IFA and by two POC IC assays. Cohen's Kappa coefficient (0.748, p < 0.0001), indicated a substantial agreement between the original and repeat serologic testing results. An almost identical high sensitivity and moderate specificity were established for the two POC IC assays. Canine serum specimens on long-term storage may still be of value for seroepidemiologic surveys investigating the exposure to E. canis.

Epidemiological study on Anaplasma phagocytophilum in cattle: Molecular prevalence and risk factors assessment in different ecological zones in Iran.

Anaplasma phagocytophilum is a tick-borne pathogen affecting humans and domestic animals worldwide. This study aimed to determine the molecular epidemiology and its associated risk factors of A. phagocytophilum infection in cattle in four ecological zones of Iran. A multi-stage stratified random sampling method was utilized during 2017-2018. A total of 1851 blood samples from 320 cattle farms were collected and examined using specific nested polymerase chain reaction (nPCR) based on the 16S rRNA gene. The overall prevalence of A. phagocytophilum was 15.5% (286/1851) by using nPCR. All four zones were A. phagocytophilum positive, the presence of A. phagocytophilum DNA was detected in eight out of nine tested provinces. Univariable analysis of risk factors indicated that climate, altitude, longitude, latitude, season, farm-type, feeding method, hygiene of the farm, tick infestation, use of acaricides by the farmer, distance from other farms, contact with wild animals, race, sex, and milk yield were significant determinants (P < 0.05) for A. phagocytophilum infection. The multivariable analysis determined that longitude, latitude, season, feeding method, and hygiene of the farm remained as significant risk factors for A. phagocytophilum infection (P < 0.05). Specific (SaTScan) cluster analysis identified two high risks and four low risks statistically significant clusters for A. phagocytophilum infection amongst the study areas (P < 0.001). Phylogenetic analysis indicated that A. phagocytophilum 16S rRNA isolates were 96-99% identical to sequences deposited in the GenBank. To the best of our knowledge, this is the first comprehensive molecular study on the epidemiology and risk factors analysis of A. phagocytophilum infection in cattle in different climatic zones of Iran. Further investigations are necessary to be performed regarding the tick vectors, reservoir animals, and the zoonotic potential of the A. phagocytophilum in the endemic region of Iran.

Conventional and Holter Electrocardiographic Assessment of Dogs Infected Naturally With Acute Canine Monocytic Ehrlichiosis.

Canine Monocytic Ehrlichiosis (CME) is a disease of worldwide distribution caused by the bacteria Ehrlichia canis, appearing primarily in hot climates due to the massive prevalence of its vector, the tick Rhipicephalus sanguineus. Previous studies have shown that dogs afflicted by CME in the chronic phase can develop infectious myocarditis, arrhythmias, and alterations in heart rate variability (HRV), but there are few studies correlating cardiac diseases with the acute phase of CME. This study aims at assessing electric cardiac alterations and HRV in the time and frequency domains during the acute phase of CME. This study assessed 22 animals divided into 2 distinct groups: the control group, comprised by 10 healthy dogs, and the sick group, comprised of 12 dogs infected naturally with ehrlichiosis which presented clinical and hematological signs compatible with the acute phase of the disease. The animals underwent conventional and Holter electrocardiographic evaluations, systolic blood pressure measurement, complete blood count and biochemical assays (urea, creatinine, alanine aminotransferase (ALT), alkaline phosphatase (AP), and gamma glutamyl transferase (GGT)). The sick group presented higher activity in the sympathetic nervous system than in the parasympathetic nervous system, manifest as a significant increase in mean heart rate and a reduction in the HRV indexes for the time and frequency domains. The frequency-domain HRV indexes presented sympathetic prevalence during the sleep and vigilance states. Sinus tachycardia was the predominant heart rhythm in 58.33% of the animals. The mean systolic blood pressure diverged between the groups and no significant arrhythmias were observed during monitoring. The serum concentrations of urea, creatinine, AP, ALT, and GGT were within the established reference values for the species. We observed no indication that there was enough time during the acute phase for the disease to evolve in a way that resulted in arrhythmias, as is common in the chronic phase, but we observed that animals in the acute phase already present reduced HRV indexes.

Rapid and sensitive detection of Anaplasma phagocytophilum using a newly developed recombinase polymerase amplification assay.

Anaplasma phagocytophilum, the bacterial pathogen responsible for tick-borne fever and human granulocytic anaplasmosis, can seriously affect the health of humans and a wide range of other mammals. In this study, we developed a recombinase polymerase amplification (RPA) assay to detect A. phagocytophilum in clinical samples. Following alignment of the relevant DNA sequences, a pair of specific primers based on the 16S rRNA gene was designed to specifically detect A. phagocytophilum. The assay was performed at a constant temperature of 38 °C for 30 min, with a final primer concentration of 0.4 µM. The specificity of the primers was confirmed when DNA from A. phagocytophilum was used as the positive control, and DNA from other related pathogens were used as the negative controls, with ddH2O acting as the blank control. The results showed that the primers did not cross-react with DNA from the other related pathogens. The assay's detection limit was 1.77 × 10-5 ng/µl, a 10 × higher sensitivity level than that determined for nested PCR. The RPA assay's performance was evaluated using 44 clinical samples, and the prevalence results for A. phagocytophilum were found to not differ significantly between the RPA assay and the nested PCR. Thus, we have developed a specific, sensitive, rapid and cost-effective RPA method, requiring only a water bath, for the detection of A. phagocytophilum. The assay should be especially useful in resource-limited areas where access to laboratory equipment is limited.

Cross-sectional survey of health management and prevalence of vector-borne diseases, endoparasites and ectoparasites in Samoan dogs.

OBJECTIVE: To determine the prevalence of selected canine vector-borne diseases (Leishmania infantum, Anaplasma spp., Ehrlichia canis, Borrelia burgdorferi and Dirofilaria immitis) and endo- and ectoparasites in Samoan dogs presenting for surgical sterilisation and to report on the general health management of the dogs. METHODS: This study was a prospective serological cross-sectional survey. Management data were obtained for 242 dogs by interview with their owners. Blood samples were collected from 237 dogs and screened for the canine vector-borne diseases using point-of-care qualitative ELISA assays. Anaplasma spp. positive samples were screened by PCR and sequenced for species identification. Rectal faecal samples were collected from 204 dogs for faecal flotation and immunofluorescent antibody tests were performed for Giardia and Cryptosporidium spp. on a subset of 93 faecal samples. The skin and coat of 221 dogs were examined for presence of ectoparasites. RESULTS: The D. immitis antigen was detected in 46.8% (111/237) of dogs. Seroprevalence of Anaplasma spp. was 8.4% (20/237); A. platys was confirmed by PCR. Prevalence of hookworm was 92.6% (185/205) and Giardia was 29.0% (27/93). Ectoparasites were detected on 210/221 (95.0%) of dogs examined and 228/242 dogs (94.2%) had previously never received any preventative medication. CONCLUSIONS: There was a very high prevalence of D. immitis, hookworm and external parasites in Samoan dogs, and prophylactic medication is rarely administered. This is the first report confirming A. platys in Samoa and the South Pacific islands. The public health implications of poor management of the dogs should be considered and investigated further.

Assessment of a quantitative 5' nuclease real-time polymerase chain reaction using groEL gene for Ehrlichia and Anaplasma species in rodents in Brazil.

New genotypes of Anaplasmataceae agents have been detected in wild carnivores, birds and deer in Brazil. The present work aimed to investigate the presence of Ehrlichia and Anaplasma species in rodents sampled in Brazil. Additionally, a newly designed quantitative 5' nuclease real-time multiplex PCR for Ehrlichia and Anaplasma spp. detection based on groEL gene amplification was designed, showing high specificity and sensitivity (10 groEL fragment copy/µL). Between 2000 and 2011, different rodent species [n=60] were trapped in 5 Brazilian biomes. Among 458 rodent spleen samples, 0.4% (2/458) and 2.4% (11/458) were positive for Ehrlichia and Anaplasma spp., respectively. Of 458 samples, 2.0% (9/458) and 1.1% (5/458) were positive for Anaplasma sp. and Ehrlichia sp., respectively, using conventional 16S rRNA PCR assays. Maximum Likelihood phylogenetic analyse based on a small region of 16S rRNA genes positioned the Anaplasma genotypes in rodents near Anaplasma phagocytophilum or Anaplasma marginale and Anaplasma odocoilei isolates. Ehrlichia genotypes were closely related to E. canis. There was a low occurrence of Anaplasma and Ehrlichia in wild and synanthropic rodents in Brazil, suggesting the circulation of new genotypes of these agents in rodents in the studied areas.

Anaplasma phagocytophilum infection in American robins and gray catbirds: an assessment of reservoir competence and disease in captive wildlife.

Anaplasma phagocytophilum (Dumler et al.) is the bacterial agent of human granulocytic anaplasmosis, an emerging infectious disease. The main vector of A. phagocytophilum in the United States is the blacklegged tick (Ixodes scapularis (Say)) and various small and medium-sized mammals are reservoirs. Previous studies indicate that birds are exposed to A. phagocytophilum; however, because no studies have directly investigated avian susceptibility, reservoir competence, and morbidity for A. phagocytophilum, uncertainty remains as to what role birds could play in its transmission ecology. In a controlled laboratory study, we tested whether two species, the American robin (Turdus migratorius (L.)) and the gray catbird (Dumetella carolinensis (L.)), can become infected with and then transmit A. phagocytophilum to feeding ticks, and whether exposed birds develop disease. Wild caught, seronegative birds (n = 10 per species) were exposed to A. phagocytophilum-infected I. scapularis nymphs (day 0). Transmission was assessed by xenodiagnosis on days 7, 14, 42, and 77; blood was assayed for bacteremia and serology. A. phagocytophilum was detected using quantitative polymerase chain reaction targeting the 16s rRNA gene. One robin infected 2 of 13 larval ticks (15%) on day 7; no other birds were found to infect feeding ticks at any time. Birds did not develop bacteremia, specific antibodies or significant illness because of exposure. Mouse controls became bacteremic, infected feeding ticks, and seroconverted. Our results suggest that these two avian species are unlikely to play a significant role in the maintenance of the agent of human granulocytic anaplasmosis and that avian serosurveys may not be a reliable indicator of A. phagocytophilum exposure.

An assessment of whole blood and fractions by nested PCR as a DNA source for diagnosing canine ehrlichiosis and anaplasmosis.

Ehrlichiosis and anaplasmosis are tick-borne diseases. Ehrlichia canis and Anaplasma platys infect mainly white cells and platelets, respectively. The main DNA source for PCR is peripheral blood, but the potential of blood cell fractions has not been extensively investigated. This study aims at assessment of whole blood (WB) and blood fractions potential in nested PCR (nPCR) to diagnose canine ehrlichiosis and anaplasmosis. The 16S rRNA gene was amplified in 71.4, 17.8, 31.57, and 30% of the WB, granulocyte (G), mononuclear cells (M), and buffy coat (BC) samples. Compared to the WB, the sensitivity of the PCR was 42.86% for the M, and BC fractions, 21.43% for the G, and 33.33% for the blood clot (C). There was fair agreement between the WB and M, BC and C, and slight with the G. Fair agreement occurred between the nPCR and morulae in the blood smear. One animal was coinfected with A. platys and E. canis. This study provided the first evidence of A. platys infection in dogs in Paraíba, Brazil, and demonstrated that WB is a better DNA source than blood fractions to detect Ehrlichia and Anaplasma by nPCR, probably because of the plasma bacterial concentration following host cell lysis.

General review of tick-borne diseases of sheep and goats world-wide.

A general review of the tick-borne diseases of sheep and goats is given, with the emphasis on those thought to be of greatest economic importance. These include babesiosis, theileriosis, cowdriosis, anaplasmosis, ehrlichiosis, Nairobi sheep diseases and tick paralysis. A commented list of tick-borne diseases and their vectors is presented. It is stressed that large gaps remain in our knowledge of the real importance in the field of many of these diseases, especially in local stock.

Evaluation of vaccination of horses as a strategy to control equine monocytic ehrlichiosis.

OBJECTIVE: To determine whether preferentially vaccinated horses were at risk for exposure to Ehrlichia risticii, whether horses with equine monocytic ehrlichiosis (EME) were likely to have been nonvaccinated, and whether clinical severity and financial costs associated with care and treatment of EME were less for vaccinated horses with EME than for nonvaccinated horses with EME. DESIGN: Cross-sectional and case-control studies. PROCEDURE: Information on usage of E risticii bacterins to control EME was collected for 2,587 horses located on 511 farms throughout New York. Each horse was tested for serum antibodies directed against E risticii. Data on efficacy of vaccination to reduce the prevalence and clinical severity of EME and monetary losses associated with EME were collected from 68 horses with EME and 132 clinically normal horses. RESULTS: A correlation was not detected between the county seropositive proportion and the proportion of horses vaccinated against EME. Among horses diagnosed for EME, median date of diagnosis was not delayed for vaccinated horses, compared with that for nonvaccinated horses. Mean cost per case was not significantly different for nonvaccinated horses, compared with that for vaccinated horses ($ 1,082 and $ 1,001, respectively). Vaccination was not associated with a reduction in prevalence or in severity of EME-related clinical signs. CLINICAL IMPLICATIONS: Administering killed E risticii bacterin once a year to control EME in New York appears to have limited success. Among horses in which EME was diagnosed, severity of illness and financial costs attributable to EME were indistinguishable for vaccinated and nonvaccinated horses.

Benefit-cost analysis of vaccination of horses as a strategy to control equine monocytic ehrlichiosis.

OBJECTIVE: To determine whether horses in New York should be vaccinated against equine monocytic ehrlichiosis (EME). DESIGN: Decision-tree analyses of data from a cross-sectional study and a case-control study. SAMPLE POPULATION: Horses in New York. PROCEDURE: Annual expected monetary loss per horse attributable to EME was calculated for vaccinated and nonvaccinated horses in New York. Because risk of being seropositive was dependent on county in which the horse was located, farm elevation, and use of each horse, decision-tree analyses were stratified by these factors. RESULTS: Annual expected monetary loss per horse attributable to EME for horses vaccinated by veterinarians ranged from $ 21 to $ 21.83/horse/y; for horses vaccinated by owners ranged from $ 10 to $ 10.83/horse/y; and for nonvaccinated horses ranged from $ 0 to $ 4.03/horse/y. Assuming 78% of vaccinated horses were protected and mean losses associated with EME included costs for horses that died, annual incidence density at which expected monetary loss for vaccinated horses was equal to that for nonvaccinated horses was 12 cases/1,000 horses/y and 25 cases/1,000 horses/y for horses vaccinated by owners or by veterinarians, respectively. CLINICAL IMPLICATIONS: Annual vaccination minimizes monetary losses attributable to EME only when the annual incidence density exceeds 12 to 25 cases/1,000 horses/y. In New York, expected monetary losses are minimized when horses are not vaccinated because of the low annual incidence density in most regions.