Severe equine asthma (SEA) is a common, debilitating lower airway inflammatory disorder of older horses. Alveolar macrophages (AMs) survey inhaled particulates from barn sources causing them to switch from an anti-inflammatory to a proinflammatory phenotype, resulting in neutrophil recruitment to the lung. This proinflammatory switch may contribute to the development and prolongation of SEA. Validated antibodies to identify the cells involved in the pathogenesis of SEA are lacking. In this study, monoclonal antibodies against CD90, CD163, and CD206 were tested for reactivity with equine leukocytes by immunocytochemistry and flow cytometry. A multi-color flow cytometric assay was developed to identify leukocytes in equine bronchoalveolar lavage fluid (BALF). Four control and 4 SEA-susceptible horses had BALF collected before and after a 48-hour moldy hay challenge. Antibodies against CD90 uniquely labeled equine neutrophils, and antibodies against CD163 and CD206 identified equine macrophages. Postchallenge AM surface expression of CD163 increased in both groups of horses, but the increase was statistically significant in only the SEA-susceptible group ( P = .02). The surface expression of CD206 on AMs increased significantly in the SEA-susceptible group ( P = .03) but was unchanged in the control group ( P = .5). Increased expression of CD163 and CD206 during exacerbation of SEA suggested an association between AM phenotype and lung inflammation. However, functions of AMs in the pathogenesis of SEA remain to be elucidated.
The Salivary Scavenger and Agglutinin (SALSA) protein is an innate immune protein with various alleged functions, including the regulation of inflammation and tissue remodeling. Transcriptomic studies of severe equine asthma (SEA) showed downregulation of the gene encoding SALSA in bronchial epithelium of asthmatic compared to non-asthmatic horses. This study aimed to characterize expression of SALSA in equine tissues by immunohistochemistry (IHC), corroborate potential differences in epithelial gene expression between asthmatic and non-asthmatic horses, and assess the structure of equine SALSA. An antibody against SALSA was validated through immunoprecipitation followed by mass spectrometry and Western blotting to recognize the equine protein. This antibody was applied to tissue microarrays (TMAs) containing 22 tissues each from four horses. A quantitative PCR assay was designed to compare gene expression for SALSA between six asthmatic and six non-asthmatic horses, before and after an asthmatic challenge, using cDNA from endoscopic bronchial biopsies as source material. The SALSA gene from bronchial cDNA samples of 10 horses, was amplified and sequenced, and translated to characterize the protein structure. Immunostaining for SALSA was detected in the mucosal surfaces of the trachea, bronchi, bronchioles, stomach, small intestine and bladder, in pancreatic and salivary gland ducts, and in uterine gland epithelium. Staining was strongest in the duodenum, and the intercalated ducts and Demilune cells of the salivary gland. SALSA was concentrated in the apical regions of the epithelial cell cytoplasm, suggestive of a secreted protein. Gene expression was significantly lower (p = 0.031) in asthmatic compared to non-asthmatic horses. Equine SALSA consisted of three to five scavenger receptor cysteine-rich (SRCR) domains, two CUB (C1r/C1s, uegf, bmp-1) domains and one Zona Pellucida domain. These domains mediate the binding of ligands involved in innate immunity. Varying numbers of SRCR domains were identified in different horses, indicating different isoforms. In summary, equine SALSA has a predilection for mucosal sites, has multiple isoforms, and has decreased expression in asthmatic horses, suggesting alterations in innate immunity in equine asthma.
Cryptosporidium is an important enteric parasite that can contribute large numbers of infectious oocysts to drinking water catchments. As a result of its resistance to disinfectants including chlorine, it has been responsible for numerous waterborne outbreaks of gastroenteritis. Wildlife and livestock play an important role in the transmission of Cryptosporidium in the environment. Studies conducted outside Australia have indicated that camels may also play a role in the transmission of zoonotic species of Cryptosporidium. Despite Australia being home to the world's largest camel herd, nothing is known about the prevalence and species of Cryptosporidium infecting camels in this country. In the present study, C. parvum was identified by PCR amplification and sequencing of a formalin-fixed intestinal tissue specimen from a one-week old dromedary camel (Camelus dromedarius). Subtyping analysis at the glycoprotein 60 (gp60) locus identified C. parvum subtype IIaA17G2R1, which is a common zoonotic subtype reported in humans and animals worldwide. Histopathological findings also confirmed the presence of large numbers of variably-sized (1-3 µm in diameter) circular basophilic protozoa - consistent with Cryptosporidium spp.- adherent to the mucosal surface and occasionally free within the lumen. Further analysis of the prevalence and species of Cryptosporidium in camel populations across Australia are essential to better understand their potential for contamination of drinking water catchments.
Alveolar macrophages (AMs) are the predominant innate immune cell in the distal respiratory tract. During inflammatory responses, AMs may be supplemented by blood monocytes, which differentiate into monocyte-derived macrophages (MDMs). Macrophages play important roles in a variety of common equine lower airway diseases, including severe equine asthma (SEA). In an experimental model, an inhaled mixture of Aspergillus fumigatus spores, lipopolysaccharide, and silica microspheres (FLS), induced SEA exacerbation in susceptible horses. However, whether equine AMs and MDMs have differing immunophenotypes and cytokine responses to FLS stimulation is unknown. To address these questions, alveolar macrophages/monocytes (AMMs) were isolated from bronchoalveolar lavage fluid and MDMs derived from blood of six healthy horses. Separately, AMMs and MDMs were cultured with and without FLS for six hours after which cell surface marker expression and cytokine production were analyzed by flow cytometry and a bead-based multiplex assay, respectively. Results showed that regardless of exposure conditions, AMMs had significantly higher surface expression of CD163 and CD206 than MDMs. Incubation with FLS induced secretion of IL-1β, IL-8, TNF-α and IFN-γ in AMMs, and IL-8, IL-10 and TNF-α in MDMs. These results suggest that AMMs have a greater proinflammatory response to in vitro FLS stimulation than MDMs, inferring differing roles in equine lung inflammation. Variability in recruitment and function of monocyte-macrophage populations warrant more detailed in vivo investigation in both homeostatic and diseased states.
BackgroundCanine vector-borne diseases have a worldwide distribution, but to the best of our knowledge, no research has been carried out to evaluate their presence on the Indian Ocean island of Mauritius. An investigation into canine vector-borne infections was conducted in dogs (n = 78) resident at an animal shelter in Port Louis, Mauritius using a combination of traditional microscopy and serological methods.MethodsTicks were manually collected from the stray dog population for identification as well as for quantifying tick burdens. Blood was also collected from each dog via either the jugular vein or the cephalic vein, and was stored in EDTA tubes. The stored blood was then used to measure PCV values, make blood smears for the identification of parasites, and used for serological testing of vector-borne disease.ResultsA total of 178 ticks were collected from 52 dogs and identified as Rhipicephalus sanguineus (175/178) or Amblyomma variegatum (3/178). Twenty-six (33%; 95% CI 23, 45) dogs were seropositive for Ehrlichia spp., and 12 (15%; 95% CI 8, 25) for Anaplasma spp., Dirofilaria antigen was detected in 14 (18%; 95% CI 10, 28), and nine (12%; 95% CI 5, 21) dogs had Hepatozoon canis gamonts observed in blood films during microscopic examination. Eleven (14%; 95% CI 7, 24) dogs were co-infected with two pathogens. Borrelia burgdorferi antibodies were not detected in any dogs.ConclusionsInfection with these pathogens had no significant effect on the packed cell volume (PCV), but high tick burdens were significantly associated with the presence of a tick-borne pathogen. This is the first study of its kind on the dog population in Mauritius and demonstrates the presence of previously undocumented canine vector-borne infections on the island. The relatively high proportion of infected dogs within the study should alert clinicians to the presence of canine vector-borne diseases on the island of Mauritius.
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