Molecular detection and characterization of Anaplasma platys and Ehrlichia canis in dogs from the Caribbean.

Anaplasma platys is a tick-transmitted rickettsial pathogen, which is known to be the etiologic agent for cyclic thrombocytopenia in its primary canine host. Infections with this pathogen are also reported in cats, cattle and people. Similarly, Ehrlichia canis is another tick-borne rickettsial pathogen responsible for canine monocytic ehrlichiosis and is also reported to cause infections in people. We describe infections in dogs with these two pathogens on the Caribbean island of Grenada, West Indies by detection using molecular methods. We utilized a 16S rRNA gene-based PCR assay to detect both Ehrlichia and Anaplasma species by screening 155 canine blood samples from asymptomatic dogs. We found 18.7 % of the dogs to be positive for A. platys and 16.8 % for E. canis. Samples that tested positive for A. platys were further assessed by sequence analysis targeting 16S rRNA, 23S rRNA, citrate synthase (gltA) and heat shock protein (groEL) genes. Phylogenetic analysis revealed high correlation of A. platys 16S rRNA and gltA gene sequences with the geographic origins, while 23S rRNA and groEL gene sequences clustered independent of the geographic origins. This study represents an important step in defining the widespread distribution of active rickettsial infections in Caribbean dogs with no apparent clinical signs, thus posing a high risk for canine health and to a lesser extent to humans, as most dogs in the Caribbean are free-roaming.

Development of a Multiplex PCR and Magnetic DNA Capture Assay for Detecting Six Species Pathogens of the Genera Anaplasma and Ehrlichia in Canine, Bovine, Caprine and Ovine Blood Samples from Grenada, West Indies.

Infections with tick-borne pathogens belonging to Anaplasma/Ehrlichia in various vertebrate hosts are a persistent problem resulting in nonspecific clinical signs during early infection. Diagnosis of single and multi-infections with these pathogens, causing diseases in companion/agricultural animals and people, remains a challenge. Traditional methods of diagnosis, such as microscopy and serology, have low sensitivity and specificity. Polymerase chain reaction (PCR) assays are widely used to detect early-phase infections, since these have high sensitivity and specificity. We report the development and validation of an assay involving PCR followed by magnetic capture method using species-specific oligonucleotides to detect six Anaplasma/Ehrlichia species pathogens in canine, bovine, caprine, and ovine blood samples. Overall, the assay application to 455 samples detected 30.1% (137/455) positives for one or more out of six screened pathogens. Single-pathogen infections were observed in 94.9% (130/137) of the positive samples, while co-infections were detected in 5.1% (7/137). Anaplasma marginale infection in cattle had the highest detection rate (34.4%), followed by canines positive for Anaplasma platys (16.4%) and Ehrlichia canis (13.9%). The assay aided in documenting the first molecular evidence for A. marginale in cattle and small ruminants and Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in the Caribbean island of Grenada.

Down syndrome on a small Caribbean island: an uphill battle to obtain services and care for affected individuals.

Most of the geographically isolated island nations in the Caribbean have small populations and low gross national product. As such, many lack important medical and community services. Difficulties are compounded when attempting to care for children with special needs and genetic disorders such as Down syndrome. International charitable organizations can help to provide much needed specialty medical care. Community associations can encourage local relationship building and education. Collaborative efforts with well-funded laboratories can help to deliver molecular characterization for individuals who have genetic disorders. With community and volunteer effort, a higher standard of care is obtainable in underserved communities.

Recurrent horizontal transfer identifies mitochondrial positive selection in a transmissible cancer.

Autonomous replication and segregation of mitochondrial DNA (mtDNA) creates the potential for evolutionary conflict driven by emergence of haplotypes under positive selection for 'selfish' traits, such as replicative advantage. However, few cases of this phenomenon arising within natural populations have been described. Here, we survey the frequency of mtDNA horizontal transfer within the canine transmissible venereal tumour (CTVT), a contagious cancer clone that occasionally acquires mtDNA from its hosts. Remarkably, one canine mtDNA haplotype, A1d1a, has repeatedly and recently colonised CTVT cells, recurrently replacing incumbent CTVT haplotypes. An A1d1a control region polymorphism predicted to influence transcription is fixed in the products of an A1d1a recombination event and occurs somatically on other CTVT mtDNA backgrounds. We present a model whereby 'selfish' positive selection acting on a regulatory variant drives repeated fixation of A1d1a within CTVT cells.

Somatic evolution and global expansion of an ancient transmissible cancer lineage.

The canine transmissible venereal tumor (CTVT) is a cancer lineage that arose several millennia ago and survives by "metastasizing" between hosts through cell transfer. The somatic mutations in this cancer record its phylogeography and evolutionary history. We constructed a time-resolved phylogeny from 546 CTVT exomes and describe the lineage's worldwide expansion. Examining variation in mutational exposure, we identify a highly context-specific mutational process that operated early in the cancer's evolution but subsequently vanished, correlate ultraviolet-light mutagenesis with tumor latitude, and describe tumors with heritable hyperactivity of an endogenous mutational process. CTVT displays little evidence of ongoing positive selection, and negative selection is detectable only in essential genes. We illustrate how long-lived clonal organisms capture changing mutagenic environments, and reveal that neutral genetic drift is the dominant feature of long-term cancer evolution.

Antimicrobial resistance patterns of commensal Escherichia coli isolated from feces of non-diarrheic dogs in Grenada, West Indies.

BACKGROUND AND AIM: There is currently no published information on the prevalence and antimicrobial susceptibility patterns of commensal Escherichia coli in dogs of Grenada origin. Monitoring antimicrobial resistance helps in the empirical selection of antibiotics. This study determined the occurrence of E. coli including the O157:H7 serotype in feces of non-diarrheic dogs of Grenada origin and the antibiotic resistance pattern of the E. coli isolates. MATERIALS AND METHODS: Fecal samples from 142 of the 144 (98.6%) dogs were culture positive for E. coli. Selection of up to three colonies from each of the 142 E. coli-positive samples yielded a total of 402 E. coli isolates, which were analyzed for the presence of non-sorbitol fermenting colonies, and O157-agglutination. RESULTS: Of the 402 E. coli isolates, 30 (7.5%) were non-sorbitol fermenters. However, none of the 402 isolates gave a positive reaction (O157:H7) to the E. coli O157:H7 latex kit. Antimicrobial susceptibility tests against 12 antibiotics revealed low resistance rates to all the tested antibiotics except for tetracycline (Te) (23.4%), cephalothin (CF) (13.2%), and ampicillin (AM) (7.7%). Thirty-nine out of the 402 (9.7%), E. coli isolates were resistant to two or more antibiotics of different classes. CONCLUSION: This is the first report of isolation and antimicrobial susceptibilities of commensal E. coli from non-diarrheic dogs in Grenada. Some of the isolates (39/402 isolates, 9.7%) were resistant to multiple antibiotics. This study showed that presently, dogs in Grenada should not be considered a reservoir for the E. coli O157:H7 serotype and for multiple antibiotic-resistant E. coli strains. Among the 402 E. coli isolates, the resistance rate to drugs other than Te, CF, and AM was very low.

Sex differences in an old adult sample with substance use disorder: A 6 months follow-up study.

Substance use disorder (SUD) is a worldwide concern that has its own particularities regarding age and sex. This study aims to assess the differences between old SUD women and men regarding socio-demographics, clinical factors and outcomes. A 6-months follow-up longitudinal study was conducted in an outpatient center, on a convenience sample of 115 SUD old adults (≥65 years old, average age of 71.57). Descriptive, bivariate, and multivariate analyses were performed. Data showed statistical significant differences between men and women related to sociodemographic variables (marital status, coexistence, criminal records and stress factors), medical and psychiatric conditions (women suffer higher rates of depression and anxiety, with worse health-related quality of life), family records (women had more presence of family psychiatric records) and SUD related parameters (men tend to use more alcohol, had an early onset, consume higher doses, report more craving and more tobacco life use while women had higher rates of prescription drugs use). At 6-month follow-up, the whole sample showed excellent rates of adherence and abstinence, without sex differences. The study points out sex differences on several sociodemographic and clinical variables, indicating their specific needs. This research could facilitate better approaches by considering a sex perspective in SUD old adults.

Cannabis consumption and psychosis or schizophrenia development.

OBJECTIVE:: Cannabis consumption produces psychopathology, in some cases psychotic episodes, which are of our interest in this work. However, the relationship between cannabis use and psychosis has not been fully elucidated. The objectives of this work are to (1) review the current state of knowledge on the association of cannabis use with the risk of the development of psychosis or psychotic symptoms in people without schizophrenia and (2) assess the consistency of the hypothesis that cannabis use is associated with increased risk of psychosis in people without schizophrenia. METHOD:: This work included research done in humans until May 2018 with the keywords 'cannabis' and 'psychosis', published in English and Spanish, in the PubMed database. RESULTS:: In all, 66 papers were analyzed, of which 23 were cohort trials and 43 were reviews. CONCLUSION:: Cannabis use doubles the risk of developing psychosis in vulnerable people. There even exists a relationship regarding the dose used and the age of first use. Gene-environment interactions that modulate the association between cannabis use and the presence of psychosis have also been described.

Comparison of intraperitoneal ropivacaine and bupivacaine for postoperative analgesia in dogs undergoing ovariohysterectomy.

OBJECTIVE: To compare postoperative analgesia following either intraperitoneal (IP) ropivacaine or bupivacaine in dogs undergoing ovariohysterectomy (OVH) in the scope of multimodal analgesia. STUDY DESIGN: Prospective, randomized, blinded clinical study. ANIMALS: A total of 45 privately owned dogs undergoing OVH, aged 37 ± 28 months and weighing 11.3 ± 4.5 kg. METHODS: Dogs were premedicated with acepromazine (0.05 mg kg-1) and morphine (0.5 mg kg-1) intramuscularly (IM). Anaesthesia was induced with alfaxalone and maintained with isoflurane in oxygen. Carprofen (4 mg kg-1) was injected subcutaneously after intubation. Dogs were randomly assigned to receive either bupivacaine (group B; 3 mg kg-1) or ropivacaine (group R; 3 mg kg-1) IP prior to complete closure of the linea alba. At 0.5, 1, 2, 4, 6 and 8 hours after extubation, sedation and postoperative pain were assessed, using the short form of the Glasgow Composite Pain scale (GCPS-SF), a dynamic interactive visual analogue scale (DIVAS), and mechanical nociceptive threshold (MNT) measurement. Rescue morphine (0.2 mg kg-1) was administered in case of ≥ 5/20 or ≥ 6/24 in the GCPS-SF and/or >40 mm in the DIVAS. Parametric data were compared using the t test; nonparametric data were analysed with the two-sample Wilcoxon test (p < 0.05). RESULTS: The GCPS-SF score was significantly higher in group R at 8 hours. There was no other significant difference regarding sedation or analgesia between the groups. Rescue analgesia was administered to 15 dogs (R: 9/22; B: 6/22), with no significant difference between the groups. MNT values decreased in both groups at all time points when compared to baseline. No adverse effects were observed. CONCLUSIONS AND CLINICAL RELEVANCE: Ropivacaine or bupivacaine IP in combination with morphine IM and carprofen SC provided comparable postoperative analgesia in dogs after OVH for 6 hours. However, the anaesthetic protocol used did not prevent the administration of rescue analgesia in 41% of animals.

Harm reduction program use, psychopathology and medical severity in patients with methadone maintenance treatment. / Uso de reducción de daños y gravedad psicopatológica y médica en pacientes en programa de mantenimiento con metadona.

Methadone maintenance programs (MMP) for opioid dependence treatment have been widely used due to their effective therapeutic outcomes. Harm reduction programs (HRP) are complementary programs for severe patients with high risk behaviors and when abstinence is not possible. This study aims to compare patients in MMP that use HRP (MMP-HRP) and patients in MMP who do not use HRP (MMP-NO HRP). The sample was composed of 143 patients (MMP-HRP = 42 vs. MMP-NO HRP = 101). An additional subanalysis was performed with patients under 45 years of age (n = 116; MMP-HRP = 38 vs. MMP-NO HRP = 78). All patients were assessed with an ad hoc socio-demographic questionnaire, EuropASI, SCID-I, and SCID-II. Results show that MMP-HRP patients were younger with more frequent use of intravenous drugs and with a high prevalence of Cluster B personality disorders. MMP-NO HRP patients had lower methadone doses compared to MMP-HRP patients and preferred to use drugs by smoked route more frequently. In the subanalysis of patients under 45, MMP-HRP patients were younger, had a higher prevalence of liver diseases, more intravenous drug use, greater severity on the drug use scale, less social and family support in the suescales of EUROP-ASI than compared to patients under 45 years in the group MMP-NO HRP. In conclusion, MMP-HRP patients are younger compared to MMP-NO HRP patients, they also receive higher doses of methadone and had more intravenous use. The above findings imply that the early onset of high risk drug use and long-term exposure to heroin have more severe outcomes such as higher comorbidities (e.g. infectious diseases, medical and psychiatric disorders), and consequently, these patients are a more vulnerable group with a worse prognosis.

Los programas de mantenimiento con metadona (PMM) para el tratamiento de la dependencia a opiáceos han demostrado elevada eficacia, siendo los más utilizados en la actualidad. La Reducción de Daños (REDAN) se refiere a tratamientos y abordajes complementarios en personas con graves dificultades para la abstinencia y conductas de riesgo. El objetivo del presente trabajo es comparar pacientes en PMM que utilizan servicios REDAN (PMM-REDAN) versus pacientes en PMM que no utilizan servicios REDAN (PMM-NO REDAN). Se incluyó un total de 143 pacientes en PMM (PMM-REDAN = 42, PMM-NO REDAN = 101) y se realizó un subanálisis de los menores de 45 años (n = 116; 38 PMM-REDAN, 78 PMM-NO REDAN). Se hizo una evaluación de datos socio-demográficos, EuropASI, SCID-I y SCID-II. Los pacientes PMM-REDAN son más jóvenes, utilizan la vía parenteral y presentan trastornos de personalidad Clúster B comórbidos a la adicción. Los PMM-NO REDAN consumen más por vía fumada y tienen dosis bajas de metadona. Los menores de 45 años PMM-REDAN son más jóvenes, tienen mayor prevalencia de enfermedades hepáticas, utilizan más la vía parenteral, tienen un consumo de drogas más grave y menos soporte socio-familiar en las subescalas EUROP-ASI que los menores de 45 años PMM-NO REDAN. En conclusión, los pacientes PMM-REDAN son más jóvenes, reciben dosis mayores de metadona y utilizan vías de mayor riesgo, hecho que implica un inicio temprano en prácticas de consumo de riesgo, mayor tiempo de exposición a la heroína, con consecuencias de mayor gravedad de la adicción, mayor comorbilidad infecciosa, médica y psiquiátrica, siendo un grupo vulnerable y con pronóstico desfavorable.

Uso de reducción de daños y gravedad psicopatológica y médica en pacientes en programa de mantenimiento con metadona / Harm reduction program use, psychopathology and medical severity in patients with methadone maintenance treatment

Los programas de mantenimiento con metadona (PMM) para el tratamiento de la dependencia a opiáceos han demostrado elevada eficacia, siendo los más utilizados en la actualidad. La Reducción de Daños (REDAN) se refiere a tratamientos y abordajes complementarios en personas con graves dificultades para la abstinencia y conductas de riesgo. El objetivo del presente trabajo es comparar pacientes en PMM que utilizan servicios REDAN (PMM-REDAN) versus pacientes en PMM que no utilizan servicios REDAN (PMM-NO REDAN). Se incluyó un total de 143 pacientes en PMM (PMM-REDAN = 42, PMM-NO REDAN = 101) y se realizó un subanálisis de los menores de 45 años (n = 116; 38 PMM-REDAN, 78 PMM-NO REDAN). Se hizo una evaluación de datos socio-demográficos, EuropASI, SCID-I y SCID-II. Los pacientes PMM-REDAN son más jóvenes, utilizan la vía parenteral y presentan trastornos de personalidad Clúster B comórbidos a la adicción. Los PMM-NO REDAN consumen más por vía fumada y tienen dosis bajas de metadona. Los menores de 45 años PMM-REDAN son más jóvenes, tienen mayor prevalencia de enfermedades hepáticas, utilizan más la vía parenteral, tienen un consumo de drogas más grave y menos soporte socio-familiar en las subescalas EUROP-ASI que los menores de 45 años PMM-NO REDAN. En conclusión, los pacientes PMM-REDAN son más jóvenes, reciben dosis mayores de metadona y utilizan vías de mayor riesgo, hecho que implica un inicio temprano en prácticas de consumo de riesgo, mayor tiempo de exposición a la heroína, con consecuencias de mayor gravedad de la adicción, mayor comorbilidad infecciosa, médica y psiquiátrica, siendo un grupo vulnerable y con pronóstico desfavorable

Methadone maintenance programs (MMP) for opioid dependence treatment have been widely used due to their effective therapeutic outcomes. Harm reduction programs (HRP) are complementary programs for severe patients with high risk behaviors and when abstinence is not possible. This study aims to compare patients in MMP that use HRP (MMP-HRP) and patients in MMP who do not use HRP (MMP-NO HRP). The sample was composed of 143 patients (MMP-HRP = 42 vs. MMP-NO HRP = 101). An additional subanalysis was performed with patients under 45 years of age (n = 116; MMP-HRP = 38 vs. MMP-NO HRP = 78). All patients were assessed with an ad hoc socio-demographic questionnaire, EuropASI, SCID-I, and SCID-II. Results show that MMP-HRP patients were younger with more frequent use of intravenous drugs and with a high prevalence of Cluster B personality disorders. MMP-NO HRP patients had lower methadone doses compared to MMP-HRP patients and preferred to use drugs by smoked route more frequently. In the subanalysis of patients under 45, MMP-HRP patients were younger, had a higher prevalence of liver diseases, more intravenous drug use, greater severity on the drug use scale, less social and family support in the suescales of EUROP-ASI than compared to patients under 45 years in the group MMP-NO HRP. In conclusion, MMP-HRP patients are younger compared to MMP-NO HRP patients, they also receive higher doses of methadone and had more intravenous use. The above findings imply that the early onset of high risk drug use and long-term exposure to heroin have more severe outcomes such as higher comorbidities (e.g. infectious diseases, medical and psychiatric disorders), and consequently, these patients are a more vulnerable group with a worse prognosis

Initial development and preliminary evaluation of a multiplex bead assay to detect antibodies to Ehrlichia canis, Anaplasma platys, and Ehrlichia chaffeensis outer membrane peptides in naturally infected dogs from Grenada, West Indies.

Tick-borne bacteria, Ehrlichia canis, Anaplasma platys, and Ehrlichia chaffeensis are significant pathogens of dogs worldwide, and coinfections of E. canis and A. platys are common in dogs on the Caribbean islands. We developed and evaluated the performance of a multiplex bead-based assay to detect antibodies to E. canis, A. platys, and E. chaffeensis peptides in dogs from Grenada, West Indies, where E. canis and A. platys infections are endemic. Peptides from outer membrane proteins of P30 of E. canis, OMP-1X of A. platys, and P28-19/P28-14 of E. chaffeensis were coupled to magnetic beads. The multiplex peptide assay detected antibodies in dogs experimentally infected with E. canis and E. chaffeensis, but not in an A. platys experimentally infected dog. In contrast, the multiplex assay and an in-house enzyme-linked immunosorbent assay (ELISA) detected A. platys antibodies in naturally infected Grenadian dogs. Following testing of 104 Grenadian canine samples, multiplex assay results had good agreement with commercially available ELISA and immunofluorescent assay for E. canis antibody-positive dogs ( K values of 0.73 and 0.84), whereas A. platys multiplex results had poor agreement with these commercial assays ( K values of -0.02 and 0.01). Prevalence of seropositive E. canis and A. platys Grenadian dogs detected by the multiplex and commercial antibody assays were similar to previous reports. Although the multiplex peptide assay performed well in detecting the seropositive status of dogs to E. canis and had good agreement with commercial assays, better antigen targets are necessary for the antibody detection of A. platys.

Mitochondrial genetic diversity, selection and recombination in a canine transmissible cancer.

Canine transmissible venereal tumour (CTVT) is a clonally transmissible cancer that originated approximately 11,000 years ago and affects dogs worldwide. Despite the clonal origin of the CTVT nuclear genome, CTVT mitochondrial genomes (mtDNAs) have been acquired by periodic capture from transient hosts. We sequenced 449 complete mtDNAs from a global population of CTVTs, and show that mtDNA horizontal transfer has occurred at least five times, delineating five tumour clades whose distributions track two millennia of dog global migration. Negative selection has operated to prevent accumulation of deleterious mutations in captured mtDNA, and recombination has caused occasional mtDNA re-assortment. These findings implicate functional mtDNA as a driver of CTVT global metastatic spread, further highlighting the important role of mtDNA in cancer evolution.

Doxycycline treatment of asymptomatic dogs seropositive for Ehrlichia canis

In Grenada, West Indies dogs are at frequent exposure to the rickettsial pathogen, Ehrlichia canis, as demonstrated by high seroprevalence rates. However, many of these seropositive dogs are clinically normal. In this study we identified clinically normal, E. canis seropositive dogs and assigned half to an antibiotic treatment group and half to a no treatment group. All dogs were evaluated for the presence of E. canis DNA by PCR on whole blood before, during and after treatment. Only one seropositive dog was also PCR+ before treatment. Our results suggest that most clinically normal, E. canis seropositive dogs in a highly endemic geographic area are not concurrently infected and thus routine treatment of clinically normal, seropositive dogs is not warranted.

Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia spp. in dogs from Grenada.

To identify the tick-borne pathogens in dogs from Grenada, we conducted a serologic survey for Ehrlichia canis in 2004 (104 dogs) and a comprehensive serologic and molecular survey for a variety of tick-borne pathogens in 2006 (73 dogs). In 2004 and 2006, 44 and 32 dogs (42.3% and 43.8%) were seropositive for E. canis, respectively. In 2006, several tick-borne pathogens were identified by serology and PCR. DNA of E. canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, and Bartonella sp. were identified in 18 (24.7%), 14 (19.2%), 5 (7%), 5 (7%), and 1 (1.4%) dogs, respectively. Six (8.2%) dogs were seropositive for Bartonella vinsonii subsp. berkhoffii. All dogs were seronegative and PCR-negative for Rickettsia spp. Coinfection with two or three pathogens was observed in eight dogs. Partial 16S rRNA E. canis and A. platys sequences were identical to sequences in GenBank. Partial 18S rRNA gene sequences from the Grenadian H. canis were identical to each other and had one possible mismatch (ambiguous base) from H. canis detected from Spain and Brazil. Grenadian B. c. vogeli sequences were identical to B. c. vogeli from Brazil and Japan. All of the detected pathogens are transmitted, or suspected to be transmitted, by Rhipicephalus sanguineus. Results of this study indicate that dogs from Grenada are infected with multiple tick-borne pathogens; therefore, tick-borne diseases should be included as differentials for dogs exhibiting thrombocytopenia, leukopenia, fever, or lethargy. One pathogen, E. canis, is also of potential public health significance.