Prevalence of Trypanosoma cruzi infection and associated histologic findings in domestic cats (Felis catus).

Trypanosoma cruzi is a zoonotic protozoan parasite transmitted by triatomines that infects a wide range of mammals. South Texas is a hotspot for triatomines, T. cruzi-infected dogs and wildlife, and local transmission to humans also occurs. However, little is known about the infection of domestic cats (Felis catus) in the United States. Given the role cats play in the ecology of T. cruzi in Mexico and South America, we hypothesized that T. cruzi infection occurs in cats from south Texas, sometimes associated with cardiac pathology. In 2017, 167 euthanized cats from a south Texas shelter were sampled across winter, spring, and summer. We collected whole blood and hearts from all cats, with additional tissues from a subset. Serum samples were screened for T. cruzi antibodies using two independent rapid immunochromatographic tests and an indirect fluorescent antibody test. Cats were considered seropositive if they were positive on at least two independent serological tests. Blood clot, heart tissue and other tissues were subjected to qPCR for parasite detection and discrete typing unit (DTU) determination. Tissues from selected seropositive or PCR-positive animals and a subset of negative animals were processed routinely for histopathology and examined by a board-certified pathologist. A total of 19 cats (11.4%) were seropositive and three cats (1.8%) - one of which was seropositive - had one or more PCR-positive tissues. Infected tissues included heart, bicep femoris muscle, sciatic nerve, esophagus, and mesentery. Genotyping of the parastite to the level of DTU showed that exclusively DTU TcI was present, despite past studies showing both TcI and TcIV in vectors of the region. Eight of 19 (42.1%) seropositive cats exhibited lymphoplasmacytic inflammation, sometimes with fibrosis, in cardiac tissue compared to 28.6% of 28 seronegative cats (P = 0.10). Domestic cats are affected hosts in the eco-epidemiology of Chagas disease. Future prospective studies are needed to understand disease progression. Veterinarians in the southern United States should consider T. cruzi in their index of suspicion in cats with exposure to vectors and undetermined cardiac abnormalities.

Epidemiology and Molecular Typing of Trypanosoma cruzi in Naturally-Infected Hound Dogs and Associated Triatomine Vectors in Texas, USA.

BACKGROUND: Trypanosoma cruzi is the etiologic agent of Chagas disease throughout the Americas. Few population-level studies have examined the epidemiology of canine infection and strain types of T. cruzi that infect canines in the USA. We conducted a cross-sectional study of T. cruzi infection in working hound dogs in south central Texas, including analysis of triatomine vectors collected within kennel environments. METHODOLOGY/PRINCIPLE FINDINGS: Paired IFA and Chagas Stat-Pak serological testing showed an overall seroprevalence of 57.6% (n = 85), with significant variation across kennels. Dog age had a marginally significant effect on seropositivity, with one year of age increase associated with a 19.6% increase in odds of being seropositive (odds ratio 95% CI 0.996-1.435; p = 0.055). PCR analyses of blood revealed 17.4% of dogs harbored parasite DNA in their blood, including both seronegative and seropositive dogs. Molecular screening of organs from opportunistically sampled seropositive dogs revealed parasite DNA in heart, uterus, and mammary tissues. Strain-typing showed parasite discrete typing units (DTU) TcI and TcIV present in dog samples, including a co-occurrence of both DTUs in two individual dogs. Bloodmeal analysis of Triatoma gerstaeckeri and Triatoma sanguisuga insects collected from the kennels revealed exclusively dog DNA. Vector infection with T. cruzi was 80.6% (n = 36), in which T. gerstaeckeri disproportionately harbored TcI (p = 0.045) and T. sanguisuga disproportionately harbored TcIV (p = 0.029). Tracing infection status across dog litters showed some seropositive offspring of seronegative dams, suggesting infection of pups from local triatomine vectors rather than congenital transmission. CONCLUSIONS/SIGNIFICANCE: Canine kennels are high-risk environments for T. cruzi transmission, in which dogs likely serve as the predominant parasite reservoir. Disease and death of working dogs from Chagas disease is associated with unmeasured yet undoubtedly significant financial consequences because working dogs are highly trained and highly valued.

Molecular and serological in-herd prevalence of Anaplasma marginale infection in Texas cattle.

Bovine anaplasmosis is an infectious, non-contagious disease caused by the rickettsial pathogen Anaplasma marginale (A. marginale). The organism has a global distribution and infects erythrocytes, resulting in anemia, jaundice, fever, abortions and death. Once infected, animals remain carriers for life. The carrier status provides immunity to clinical disease, but is problematic if infected and naïve cattle are comingled. Knowledge of infection prevalence and spatial distribution is important in disease management. The objective of this study was to assess A. marginale infection in-herd prevalence in Texas cattle using both molecular and serological methods. Blood samples from 11 cattle herds within Texas were collected and analyzed by reverse transcription quantitative real-time PCR (RT-qPCR) and a commercial competitive enzyme-linked immunosorbent assay (cELISA). Samples from experimentally infected animals were also analyzed and RT-qPCR detected A. marginale infection up to 15 days before cELISA, providing empirical data to support the interpretation of herd prevalence results. Herds with high prevalence were located in the north Texas Rolling Plains and west Trans-Pecos Desert, with RT-qPCR prevalence as high as 82% and cELISA prevalence as high as 88%. Overall prevalence was significantly higher in cattle in north and west Texas compared to cattle in east Texas (p<0.0001 for prevalence based on both RT-qPCR and cELISA). The overall RT-qPCR and cELISA results exhibited 90% agreement (kappa=0.79) and provide the first A. marginale infection prevalence study for Texas cattle using two diagnostic methods. Since cattle are the most important reservoir host for A. marginale and can serve as a source of infection for tick and mechanical transmission, information on infection prevalence is beneficial in the development of prevention and control strategies.

Seroprevalence of Anaplasma marginale in Texas cattle.

To our knowledge the seroprevalence of Anaplasma marginale in Texas has not been reported. The objective of this study was to estimate the point seroprevalence and spatial distribution of Texas cattle persistently infected with A. marginale. This was a cross-sectional observational study examining serum collected from 12,000 adult cattle marketed in 23 selected Texas auction markets during the second week of July 2011. A random subset of those cattle comprising 1835 individuals was evaluated for persistent infection with A. marginale using a commercial cELISA for antibody detection. The pooled apparent seroprevalence for cattle tested at auction markets across the state was 15.02% (95% CI: 11.02-19.53%), with markets in the western portion of the state demonstrating prevalence ⇒ 30%. The winter tick, Dermacentor albipictus is involved in the biological transfer of A. marginale and is prevalent in west Texas. Producers in endemic and non-endemic areas should be encouraged to determine the infection status of replacement cattle in order to implement effective management strategies for the control bovine anaplasmosis.

Validation of an improved competitive enzyme-linked immunosorbent assay to detect Equine arteritis virus antibody.

The objective of the present study was to validate a previously described competitive enzyme-linked immunosorbent assay (cELISA) to detect antibody to Equine arteritis virus (EAV) based on GP5-specific nonneutralizing monoclonal antibody (mAb) 17B7(9) using the World Organization for Animal Health (OIE)-recommended protocol, which includes the following 5 in-house analyses. 1) The assay was calibrated with the OIE-designated reference serum panel for EAV; 2) repeatability was evaluated within and between assay runs; 3) analytical specificity was evaluated using sera specific to related viruses; 4) analytical sensitivity was evaluated with sera from horses vaccinated with an EAV modified live virus (MLV) vaccine; and 5) the duration of cELISA antibody detection following EAV vaccination was determined. The positive cELISA cutoff of ≥35% inhibition (%I) was confirmed by receiver operating characteristic plot analysis. Analytical sensitivity of the cELISA was comparable to the serum neutralization (SN) assay in that it detected EAV-specific antibody as early as 8 days postvaccination. The duration of EAV-specific antibody detected by cELISA was over 5 years after the last vaccination. This cELISA could detect EAV-specific antibody in serum samples collected from horses infected with various EAV strains. In the field trial performed by American Association of Veterinary Laboratory Diagnosticians-accredited state laboratories and OIE laboratory, the diagnostic specificity of the cELISA was 99.5% and the diagnostic sensitivity was 98.2%. The data using various serum panels also had consistently significant positive correlation between SN titers and cELISA %I results. The results further confirm that the EAV antibody cELISA is a reliable, simple alternative to the SN assay for detecting EAV-specific antibodies in equine sera.

Development and performance evaluation of calf diarrhea pathogen nucleic acid purification and detection workflow.

Calf diarrhea (scours) is a primary cause of illness and death in young calves. Significant economic losses associated with this disease include morbidity, mortality, and direct cost of treatment. Multiple pathogens are responsible for infectious diarrhea, including, but not limited to, Bovine coronavirus (BCV), bovine Rotavirus A (BRV), and Cryptosporidium spp. Identification and isolation of carrier calves are essential for disease management. Texas Veterinary Medical Diagnostic Laboratory current methods for calf diarrhea pathogen identification include electron microscopy (EM) for BCV and BRV and a direct fluorescent antibody test (DFAT) for organism detection of Cryptosporidium spp. A workflow was developed consisting of an optimized fecal nucleic acid purification and multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) for single tube concurrent detection of BCV, BRV, and Cryptosporidium spp., and an internal control to monitor nucleic acid purification efficacy and PCR reagent functionality. In "spike-in" experiments using serial dilutions of each pathogen, the analytical sensitivity was determined to be <10 TCID(50)/ml for BCV and BRV, and <20 oocysts for Cryptosporidium spp. Analytical specificity was confirmed using Canine and Feline coronavirus, Giardia spp., and noninfected bovine purified nucleic acid. Diagnostic sensitivity was ≥98% for all pathogens when compared with respective traditional methods. The results demonstrate that the newly developed assay can purify and subsequently detect BCV, BRV, and Cryptosporidium spp. concurrently in a single PCR, enabling simplified and streamlined calf diarrhea pathogen identification.