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.

Creating the Next Generation of Evidence-Based Veterinary Practitioners and Researchers: What are the Options for Globally Diverse Veterinary Curricula?

Veterinary educators strive to prepare graduates for a variety of career options with the skills and knowledge to use and contribute to research as part of their lifelong practice of evidence-based veterinary medicine (EBVM). In the veterinary curriculum, students should receive a grounding in research and EBVM, as well as have the opportunity to consider research as a career. Seeing a lack of a cohesive body of information that identified the options and the challenges inherent to embedding such training in veterinary curricula, an international group was formed with the goal of synthesizing evidence to help curriculum designers, course leaders, and teachers implement educational approaches that will inspire future researchers and produce evidence-based practitioners. This article presents a literature review of the rationale, issues, and options for research and EBVM in veterinary curricula. Additionally, semi-structured interviews were conducted with 11 key stakeholders across the eight Council for International Veterinary Medical Education (CIVME) regions. Emergent themes from the literature and interviews for including research and EBVM skills into the curriculum included societal need, career development, and skills important to clinical professional life. Approaches included compulsory as well as optional learning opportunities. Barriers to incorporating these skills into the curriculum were grouped into student and faculty-/staff-related issues, time constraints in the curriculum, and financial barriers. Having motivated faculty and contextualizing the teaching were considered important to engage students. The information has been summarized in an online "toolbox" that is freely available for educators to inform curriculum development.

Metagenomic analysis of Aedes aegypti and Culex quinquefasciatus mosquitoes from Grenada, West Indies.

The mosquitoes Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) and Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) are two major vectors of arthropod-borne pathogens in Grenada, West Indies. As conventional vector control methods present many challenges, alternatives are urgently needed. Manipulation of mosquito microbiota is emerging as a field for the development of vector control strategies. Critical to this vector control approach is knowledge of the microbiota of these mosquitoes and finding candidate microorganisms that are common to the vectors with properties that could be used in microbiota modification studies. Results showed that bacteria genera including Asaia, Escherichia, Pantoea, Pseudomonas, and Serratia are common to both major arboviral vectors in Grenada and have previously been shown to be good candidates for transgenetic studies. Also, for the first time, the presence of Grenada mosquito rhabdovirus 1 is reported in C. quinquefasciatus.

High prevalence of Phasi Charoen-like virus from wild-caught Aedes aegypti in Grenada, W.I. as revealed by metagenomic analysis.

Arboviruses cause diseases of significant global health concerns. Interactions between mosquitoes and their microbiota as well as the important role of this interaction in the mosquito's capacity to harbor and transmit pathogens have emerged as important fields of research. Aedes aegypti is one of the most abundant mosquitoes in many geographic locations, a vector capable of transmitting a number of arboviruses such as dengue and Zika. Currently, there are few studies on the metavirome of this mosquito particularly in the Americas. This study analyzes the metavirome of A. aegypti from Grenada, a Caribbean nation with tropical weather, abundant A. aegypti, and both endemic and arboviral pathogens transmitted by this mosquito. Between January and December 2018, 1152 mosquitoes were collected from six semi-rural locations near houses in St. George Parish, Grenada, by weekly trapping using BG-Sentinel traps. From these, 300 A. aegypti were selected for analysis. The metavirome was analyzed using the Illumina HiSeq 1500 for deep sequencing. The generation sequencing library construction protocol used was NuGEN Universal RNA with an average read length of 125 bp. Reads were mapped to the A. aegypti assembly. Non-mosquito reads were analyzed using the tools FastViromeExplorer. The NCBI total virus, RNA virus, and eukaryotic virus databases were used as references. The metagenomic comparison analysis showed that the most abundant virus-related reads among all databases and assemblies was Phasi Charoen-like virus. The Phasi Charoen-like virus results are in agreement to other studies in America, Asia and Australia. Further studies using wild-caught mosquitoes is needed to assess the impact of this insect-specific virus on the A. aegypti lifecycle and vector capacity.

Phylogenetic relationships and diversity of bat-associated Leptospira and the histopathological evaluation of these infections in bats from Grenada, West Indies.

Bats can harbor zoonotic pathogens, but their status as reservoir hosts for Leptospira bacteria is unclear. During 2015-2017, kidneys from 47 of 173 bats captured in Grenada, West Indies, tested PCR-positive for Leptospira. Sequence analysis of the Leptospira rpoB gene from 31 of the positive samples showed 87-91% similarity to known Leptospira species. Pairwise and phylogenetic analysis of sequences indicate that bats from Grenada harbor as many as eight undescribed Leptospira genotypes that are most similar to known pathogenic Leptospira, including known zoonotic serovars. Warthin-Starry staining revealed leptospiral organisms colonizing the renal tubules in 70% of the PCR-positive bats examined. Mild inflammatory lesions in liver and kidney observed in some bats were not significantly correlated with renal Leptospira PCR-positivity. Our findings suggest that Grenada bats are asymptomatically infected with novel and diverse Leptospira genotypes phylogenetically related to known pathogenic strains, supporting the hypothesis that bats may be reservoirs for zoonotic Leptospira.

STREPTOCOCCUS PHOCAE IN MARINE MAMMALS OF NORTHEASTERN PACIFIC AND ARCTIC CANADA: A RETROSPECTIVE ANALYSIS OF 85 POSTMORTEM INVESTIGATIONS.

: Streptococcus phocae is a pathogen of marine mammals, although its pathogenicity remains poorly understood. Recovery of this bacterium from asymptomatic carriers suggests that it is an opportunistic pathogen. We investigated the role of S. phocae in naturally occurring disease and its significance as a pathogen based on postmortem investigations. Between 2007 and 2012, 1,696 whole carcasses, tissue samples, or both were submitted from the northeastern Pacific and Arctic Canada for diagnostic testing. Streptococcus phocae was cultured from phocids ( n=66), otariids ( n=12), harbor porpoises ( Phocoena phocoena; n=5), and sea otters ( Enhydra lutris; n=2). Pathologic manifestations of S. phocae-associated disease included localized, as well as systemic, inflammatory lesions with common findings of suppurative bronchopneumonia ( n=17) and bacteremia ( n=27). Lung lesions were frequently culture-positive for S. phocae, suggesting commensal colonization of the oropharynx with subsequent opportunistic infection of the respiratory tract during tissue injury, coinfection, immunosuppression, or other debilitating conditions. The presence of a positive spleen culture, and interpretations at necropsy and histopathology, were used to determine the presence of S. phocae bacteremia. Less frequent lesions that were culture positive for S. phocae included abscesses ( n=9), meningitis ( n=7), and cellulitis ( n=1). The majority of cases with S. phocae lesions featured pre-existing conditions that presumably contributed to some degree of debilitation or immunosuppression, including emaciation ( n=29), liver mercury accumulation ( n=29), trauma ( n=22), severe pulmonary or cardiovascular nematodiasis ( n=9), concurrent bacterial or viral infections ( n=8), or sarcocystosis ( n=6). These findings suggest that S. phocae could be characterized as an opportunistic pathogen, associated with debilitating conditions in stranded and rehabilitating marine mammals. Wildlife investigators can use these results to draw more definitive conclusions regarding positive S. phocae cultures during postmortem studies in marine mammals.

Campylobacter Species Isolated from Pigs in Grenada Exhibited Novel Clones: Genotypes and Antimicrobial Resistance Profiles of Sequence Types.

Infections caused by Campylobacter species pose a severe threat to public health worldwide. However, in Grenada, the occurrence and characteristics of Campylobacter in food animals, including pigs, remain mostly unknown. In this study, we identified the sequence types (STs) of Campylobacter from young healthy pigs in Grenada and compared the results with previous studies in Grenada and other countries. Antimicrobial resistance patterns and diversity of the Campylobacter clones were evaluated. Ninety-nine Campylobacter isolates (97 Campylobacter coli and 2 Campylobacter jejuni) were analyzed by multilocus sequence typing. Eighteen previously reported STs and 13 novel STs were identified. Of the 18 previously reported STs, eight STs (ST-854, -887, -1068, -1096, -1445, -1446, 1556, and -1579) have been associated with human gastroenteritis in different geographical regions. Among these 18 previously reported STs, ST-1428, -1096, -1450, and -1058 predominated and accounted for 18.2%, 14.1%, 11.1%, and 8.1% of all isolates, respectively. Of the 13 novel STs, ST-7675 predominated and accounted for 20% (4 of 20 isolates), followed by ST-7678, -7682, and -7691, each accounting for 10% (2 of 20 isolates). Antimicrobial resistance testing using Epsilometer test revealed a low resistance rate (1-3%) of all C. coli/jejuni STs to all antimicrobials except for tetracycline (1-10.1%). Some of the C. coli STs (13 STs, 24/99 isolates, 24.2%) were resistant to multiple antimicrobials. This is the first report on antimicrobial resistance and multidrug resistance patterns associated with Campylobacter STs recovered from swine in Grenada. This study showed that pigs in Grenada are not major reservoirs for STs of C. coli and C. jejuni that are associated with human gastroenteritis worldwide.

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.

A survey of canine heartworm awareness in Grenada, West Indies.

Heartworm is a common parasite of dogs in Grenada, West Indies, due to the tropical climate and large number of mosquitoes. Because Grenada is a developing country and a small island, resources and education on heartworm are limited. In an effort to raise awareness of canine heartworm and the preventive measures available, observations and surveys were performed to determine the following: (a) heartworm knowledge among Grenadian dog caregivers seeking veterinary care at the Grenada Society for the Prevention of Cruelty to Animals (GSPCA), (b) dissemination of heartworm information by GSPCA staff to Grenadian dog caregivers, and (c) reasons Grenadians are not administering heartworm prevention to their dogs. Results indicated a marked deficiency in both heartworm knowledge among Grenadian dog caregivers and heartworm knowledge acquisition and dissemination. Lack of knowledge was also cited as a major reason Grenadian dog caregivers were not using heartworm prevention. These results are currently being utilized to modify heartworm education practices at the GSPCA by incorporating Grenada-specific educational tools, with the ultimate goal of reducing illness and death by canine heartworm.

Influence of land use and climate on Salmonella carrier status in the small Indian mongoose (Herpestes auropunctatus) in Grenada, West Indies.

Invasive mammals can be important reservoirs for human pathogens. A recent study showed that 12% of mongooses carried Salmonella spp. in their large intestines. We investigated whether anthropogenic, environmental and climatic variables predicted Salmonella status in mongooses (Herpestes auropunctatus) in Grenada. Using multivariate logistic regression and contingency table analysis, we found that increased human density, decreased distance from roads, and low monthly precipitation were associated with increased probability of Salmonella carriage. Areas with higher human density likely support a higher abundance of mongooses because of greater food availability. These areas also are a likely source for infection to mongooses due to high densities of livestock and rodents shedding Salmonella. The higher probability of Salmonella carriage in mongooses during drier months and closer to roadsides is likely due to water drainage patterns and limited water availability. Although the overall prevalence of Salmonella in mongooses was moderate, the strong patterns of ecologic correlates, combined with the high density of mongooses throughout Grenada suggest that the small Indian mongoose could be a useful sentinel for Salmonella surveillance. Its affinity for human-associated habitats suggests that the small Indian mongoose is also a risk factor in the maintenance and possible spread of Salmonella species to humans and livestock in Grenada.

Early results with the LARIAT device for left atrial appendage exclusion in patients with atrial fibrillation at high risk for stroke and anticoagulation.

OBJECTIVES: To evaluate early outcomes of left atrial appendage (LAA) closure via a percutaneous LAA ligation approach with the SentreHeart LARIAT(™) snare device. BACKGROUND: Atrial fibrillation increases the risk of stroke 4-5 fold, which can have devastating outcomes. Exclusion of the LAA is believed to decrease the risk of embolic stroke. METHODS: Twenty-seven patients with atrial fibrillation, a high risk of stroke, and contraindication or intolerance for anticoagulation therapy underwent percutaneous ligation of the LAA with the LARIAT device. Initial LAA closure was confirmed with TEE and contrast fluoroscopy. RESULTS: The acute procedural success was 92.6%. One patient sustained a perforation of the LAA and was treated conservatively. The patient underwent LAA closure surgically the next day. In one patient the attempt to advance the LARIAT over the LAA was unsuccessful. Patients were followed for a mean of 4 months. Preserved LAA closure was confirmed with a 45 day follow-up TEE in 22 of 25 patients completing the procedure. Peri-operative complications included three cases of pericarditis and one case of a periprocedural CVA due to thrombus formation on the transseptal sheath. During follow-up, there was one stroke thought to be noncardioembolic and one pleural effusion. There were no deaths. CONCLUSIONS: These results show that percutaneous LAA exclusion can be achieved successfully with an acceptable rate of periprocedural and short-term complications. Further studies and longer follow-up are needed to determine whether LAA exclusion lowers the long-term risk of thromboembolic events in patients with AF and contraindications to anticoagulation.

Prevalence and antimicrobial susceptibility of Salmonella spp. in small Indian mongooses (Herpestes auropunctatus) in Grenada, West Indies.

Intestinal samples from 156 small Indian mongooses (Herpestes auropunctatus) collected island-wide in Grenada from April 2011 to March 2013 were examined for the presence of Salmonella enterica spp. Nineteen (12%) mongooses were culture-positive for S. enterica spp. of which five serotypes were identified. Salmonella javiana and S. Montevideo were the most commonly isolated serotypes. The other serotypes isolated were S. Rubislaw, S. Panama and S. Arechavaleta. All isolates were susceptible to amoxicillin-clavulanic acid, ampicillin, cefotaxime, ceftazidime, ciprofloxacin, enrofloxacin, gentamicin, nalidixic acid, imipenem and trimethoprim-sulfamethoxazole. One isolate (S. Montevideo) showed resistance to tetracycline and intermediate resistance to streptomycin. The five isolated Salmonella serotypes are potential human pathogens suggesting that the mongoose may play a role in the epidemiology of human salmonellosis in Grenada.

Genotypes, Antibiotic Resistance, and ST-8 Genetic Clone in Campylobacter Isolates from Sheep and Goats in Grenada.

Rectal swabs from 155 sheep and 252 goats from Grenada were evaluated to determine the prevalence of Campylobacter spp., antibiotic resistance, and multilocus sequence types. Fifteen Campylobacter isolates were obtained (14 C. jejuni and 1 C. coli). The prevalence (3.7%) did not differ significantly between sheep (4.5%) and goats (3.2%). Among the seven antimicrobials tested, resistance was only detected for tetracycline (30.8%) and metronidazole (38.5%). Campylobacter isolates showed no significant difference between sheep and goats for type of antimicrobial resistance or percent of resistant isolates. Twelve of the isolates were successfully genotyped consisting of four recognized clonal complexes and three novel sequence types. Importantly, one isolate from one goat was identified as the C. jejuni sequence type-8, a zoonotic and tetracycline-resistant clone reported to be a highly virulent clone associated with ovine abortion in the USA. Although most samples were from comingled sheep and goat production units, there were no shared sequence types between these two host species. None of the sequence types identified in this study have previously been reported in poultry in Grenada, suggesting sheep- and goat-specific Campylobacter clones in Grenada. This is the first report of genotyping of Campylobacter isolates from sheep and goats in the Eastern Caribbean.

Aortic annulus measurement and relevance to successful transcatheter aortic valve replacement: a new technique using 3D TEE.

BACKGROUND: There are limitations of using 2D imaging to accurately size the aortic annulus. This article highlights the limitations of 2D technology and proposes a new 3D TEE method for sizing of the aortic annulus. METHODS: Three-dimensional echocardiography with the method described can identify the "true" aortic annulus with precision. A 3D dataset of the aortic annulus is collected. The 3D quantification program is then activated. The three multiplanar reconstruction planes (the aortic short-axis, long-axis, and coronal planes) are aligned during systole, to obtain the 3 nadirs of the sinuses of Valsalva simultaneously in the short-axis MPR. Rotating the sagittal and coronal planes to identify the hinge points is necessary to measure the diameter of the "true" annulus. The "true" aortic annulus is defined by the blood-tissue interface in the aortic short-axis MPR. RESULTS: This method has been prospectively validated with surgical AVR sizing in a small study of 10 patients. During surgical AVR, the annulus was directly measured using a sizer. This served as a gold standard for comparison. The annulus was measured using the method described in a blinded fashion by the cardiac anesthesiologist and then confirmed offline by a blinded echocardiographer. In this small study, the Pearson correlation coefficient was 0.9833 with 23.4 and 23.22 mm being the mean measurements with the 2 methods. DISCUSSION: The method described in this article is complementary to multidetector computed tomography and provides a real-time measurement of the annulus during the TAVR procedure without need for radiographic contrast.

MLST genotypes and antibiotic resistance of Campylobacter spp. isolated from poultry in Grenada.

This study determined whether multilocus sequence types (MLST) of Campylobacter from poultry in 2 farms in Grenada, West Indies, differed by farm, antimicrobial resistance and farm antibiotic use. Farm A used fluoroquinolones in the water and Farm B used tetracyclines. The E-test was used to determine resistance of isolates to seven antibiotics. PCR of the IpxA gene confirmed species and MLST was used to characterize 38 isolates. All isolates were either C. jejuni or C. coli. Farm antibiotic use directly correlated with antimicrobial resistance of Campylobacter isolates. Almost 80% of the isolates from Farm A were fluoroquinolone resistant and 17.9% of the isolates from Farm B were fluoroquinolone resistant. All Campylobacter isolates from Farm A were tetracycline sensitive, whereas 35.7% of isolates from Farm B were tetracycline resistant. Six previously recognized sequence types (STs) and 2 novel STs were identified. Previously recognized STs were those overwhelmingly reported from poultry and humans globally. Isolates with the same ST did not always have the same antibiotic resistance profile. There was little ST overlap between the farms suggesting that within-farm transmission of Campylobacter genotypes may dominate. MLST typing was useful for tracking Campylobacter spp. among poultry units and can help elucidate Campylobacter host-species population structure and its relevance to human health.

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.

Translocation of the B cell receptor to lipid rafts is inhibited in B cells from BLV-infected, persistent lymphocytosis cattle.

Bovine leukemia virus (BLV) infection causes a significant polyclonal expansion of CD5(+), IgM+ B lymphocytes known as persistent lymphocytosis (PL) in approximately 30% of infected cattle. There is evidence that this expanded B cell population has altered signaling, and resistance to apoptosis has been proposed as one mechanism of B cell expansion. In human and murine B cells, antigen binding initiates movement of the B cell receptor (BCR) into membrane microdomains enriched in sphingolipids and cholesterol, termed lipid rafts. Lipid rafts include members of the Src-family kinases and exclude certain phosphatases. Inclusion of the BCR into lipid rafts plays an important role in regulation of early signaling events and subsequent antigen internalization. Viral proteins may also influence signaling events in lipid rafts. Here we demonstrate that the largely CD5(+) B cell population in PL cattle has different mobilization and internalization of the BCR when compared to the largely CD5-negative B cells in BLV-negative cattle. Unlike B cells from BLV-negative cattle, the BCR in B cells of BLV-infected, PL cattle resists movement into lipid rafts upon stimulation and is only weakly internalized. Expression of viral proteins as determined by detection of the BLV transmembrane (TM) envelope glycoprotein gp30 did not alter these events in cells from PL cattle. This exclusion of the BCR from lipid rafts may, in part, explain signaling differences seen between B cells of BLV-infected, PL, and BLV-negative cattle and the resistance to apoptosis speculated to contribute to persistent lymphocytosis.

Rhodococcus equi secreted antigens are immunogenic and stimulate a type 1 recall response in the lungs of horses immune to R. equi infection.

Rhodococcus equi is an opportunistic pathogen in immunocompromised humans and an important primary pathogen in young horses. Although R. equi infection can produce life-threatening pyogranulomatous pneumonia, most foals develop a protective immune response that lasts throughout life. The antigen targets of this protective response are currently unknown; however, Mycobacterium tuberculosis is a closely related intracellular pathogen and provides a model system. Based on previous studies of M. tuberculosis protective antigens released into culture filtrate supernatant (CFS), a bacterial growth system was developed for obtaining R. equi CFS antigens. Potential immunogens for prevention of equine rhodococcal pneumonia were identified by using immunoblots. The 48-h CFS contained five virulence-associated protein bands that migrated between 12 and 24 kDa and were recognized by sera from R. equi-infected foals and immune adult horses. Notably, the CFS contained the previously characterized proteins VapC, VapD, and VapE, which are encoded by genes on the R. equi virulence plasmid. R. equi CFS was also examined for the ability to stimulate a type 1-like memory response in immune horses. Three adult horses were challenged with virulent R. equi, and cells from the bronchoalveolar lavage fluid were recovered before and 1 week after challenge. In vitro stimulation of pulmonary T-lymphocytes with R. equi CFS resulted in significant proliferation and a significant increase in gamma interferon mRNA expression 1 week after challenge. These results were consistent with a memory effector response in immune adult horses and provide evidence that R. equi CFS proteins are antigen targets in the immunoprotective response against R. equi infection.

Clearance of virulent but not avirulent Rhodococcus equi from the lungs of adult horses is associated with intracytoplasmic gamma interferon production by CD4+ and CD8+ T lymphocytes.

Rhodococcus equi is a gram-positive bacterium that infects alveolar macrophages and causes rhodococcal pneumonia in horses and humans. The virulence plasmid of R. equi appears to be required for both pathogenicity in the horse and the induction of protective immunity. An understanding of the mechanisms by which virulent R. equi circumvents protective host responses and by which bacteria are ultimately cleared is important for development of an effective vaccine. Six adult horses were challenged with either virulent R. equi or an avirulent, plasmid-cured derivative. By using a flow cytometric method for intracytoplasmic detection of gamma interferon (IFN-gamma) in equine bronchoalveolar lavage fluid (BALF) cells, clearance of the virulent strain was shown to be associated with increased numbers of pulmonary CD4(+) and CD8(+) T lymphocytes producing IFN-gamma. There was no change in IFN-gamma-positive cells in peripheral blood, suggesting that a type 1 recall response at the site of challenge was protective. The plasmid-cured strain of R. equi was cleared in horses without a significant increase in IFN-gamma-producing T lymphocytes in BALF. In contrast to these data, a previous report in foals suggested an immunomodulating role for R. equi virulence plasmid-encoded products in downregulating IFN-gamma expression by equine CD4(+) T lymphocytes. Intracytoplasmic detection of IFN-gamma provides a method to better determine whether modulation of macrophage-activating cytokines by virulent strains occurs uniquely in neonates and contributes to their susceptibility to rhodococcal pneumonia.