Performance of a microfluidic immunofluorescence assay kit for equine influenza virus antigen detection.

Equine influenza virus (EIV) infection is one of the most important respiratory diseases in the equine industry around the world. Rapid diagnosis, facilitated by point-of-care testing, is essential to implement movement restrictions and control disease outbreaks. This study evaluated a microfluidic immunofluorescence assay kit, which detects influenza virus and SARS-CoV-2 antigens in human specimens with a 12 min turnaround time, for its potential use in detecting EIV. The microfluidic immunofluorescence assay kit succeeded in detecting 11 EIV strains. Using the real-time reverse transcription polymerase chain reaction as a reference assay, the microfluidic immunofluorescence assay kit showed a sensitivity of 60.7% when evaluating nasopharyngeal swab samples of three horses experimentally infected with EIV. Comparing with the other two rapid antigen detection kits based on immunochromatography and silver amplification immunochromatography, the microfluidic immunofluorescence assay kit exhibited higher sensitivity than the former assay (53.6%) and the same sensitivity as the latter (60.7%). The microfluidic immunofluorescence assay kit did not detect nine non-EIV viruses including one equine coronavirus strain and seven bacteria, suggesting a high specificity for EIV antigens. Similar to other rapid antigen detection kits, the microfluidic immunofluorescence assay kit could be an effective diagnostic tool to detect EIV in the field.

Double immunofluorescence staining of whole-mount small intestinal mucosa samples as a tool for characterization of three-dimensional paratuberculosis granulomas.

Bovine paratuberculosis (PTB) is a chronic granulomatous enteritis, caused by Mycobacterium avium subsp. paratuberculosis (Map). The progression of PTB from subclinical to the clinical stage of the disease is determined locally at the level of the granuloma, a host defence hallmark against mycobacterial infection. Therefore, in-depth characterization of distinct cell populations controlling granuloma formation is critical to understanding PTB progression. Confocal laser scanning microscopy (CLSM) has been extensively used to visualize two or more proteins of interest concomitantly within a variety of cellular structures. As such, it is an invaluable tool for the correct identification and characterization of different cell populations. In this study, a novel approach, CLSM of whole-mount small intestinal mucosa samples, is used to characterize three-dimensional (3-D) paratuberculosis granulomas and epithelioid macrophages. Detailed optimized procedures to perform CLSM in whole mount small intestinal mucosa samples and also in formalin fixed paraffin embedded (FFPE) intestinal tissue sections of Holstein Friesian cows presenting different types of PTB-associated histological lesions are described.

Prospective serological and molecular cross-sectional study focusing on Bartonella and other blood-borne organisms in cats from Catalonia (Spain).

BACKGROUND: There is limited clinical or epidemiological knowledge regarding Bartonella infection in cats, and no serological studies have compared the presence of antibodies against different Bartonella species. Moreover, there are limited feline Bartonella studies investigating co-infections with other vector-borne pathogens and the associated risk factors. Therefore, the objective of this study was to investigate Bartonella spp. infections and co-infections with other pathogens in cats from Barcelona (Spain) based on serological and/or molecular techniques and to determine associated risk factors. METHODS: We studied colony and owned cats (n = 135). Sera were tested for Bartonella henselae-, Bartonella quintana-, and Bartonella koehlerae-specific antibodies using endpoint in-house immunofluorescence antibody assays. Bartonella real-time PCR (qPCR) and conventional PCR (cPCR) were performed. In addition, cPCR followed by DNA sequencing was performed for other pathogenic organisms (Anaplasma, Babesia, Cytauxzoon, Ehrlichia, Hepatozoon, hemotropic Mycoplasma, and Theileria spp.). RESULTS: From 135 cats studied, 80.7% were seroreactive against at least one Bartonella species. Bartonella quintana, B. koehlerae, and B. henselae seroreactivity was 67.4, 77.0, and 80.7%, respectively. Substantial to almost perfect serological agreement was found between the three Bartonella species. Colony cats were more likely to be Bartonella spp.-seroreactive than owned cats. Moreover, cats aged ≤ 2 years were more likely to be Bartonella spp.-seroreactive. Bartonella spp. DNA was detected in the blood of 11.9% (n = 16) of cats. Cats were infected with B. henselae (n = 12), B. clarridgeiae (n = 3), and B. koehlerae (n = 1). Mycoplasma spp. DNA was amplified from 14% (n = 19) of cat blood specimens. Cats were infected with Mycoplasma haemofelis (n = 8), Candidatus M. haemominutum (n = 6), Candidatus Mycoplasma turicensis (n = 4), and Mycoplasma wenyonii (n = 1). Anaplasma, Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria spp. DNA was not amplified from any blood sample. Of the 16 Bartonella spp.-infected cats based on PCR results, six (37%) were co-infected with Mycoplasma spp. CONCLUSIONS: Bartonella spp. and hemoplasma infections are prevalent in cats from the Barcelona area, whereas infection with Anaplasma spp., Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria infections were not detected. Co-infection with hemotropic Mycoplasma appears to be common in Bartonella-infected cats. To our knowledge, this study is the first to document M. wenyonii is infection in cats.

Prevalence of autoantibodies that bind to kidney tissues in cats and association risk with antibodies to feline viral rhinotracheitis, calicivirus, and panleukopenia.

BACKGROUND: The feline viral rhinotracheitis, calicivirus, and panleukopenia (FVRCP) vaccine, prepared from viruses grown in the Crandell-Rees feline kidney cell line, can induce antibodies to cross-react with feline kidney tissues. OBJECTIVES: This study surveyed the prevalence of autoantibodies to feline kidney tissues and their association with the frequency of FVRCP vaccination. METHODS: Serum samples and kidneys were collected from 156 live and 26 cadaveric cats. Antibodies that bind to kidney tissues and antibodies to the FVRCP antigen were determined by enzyme-linked immunosorbent assay (ELISA), and kidney-bound antibody patterns were investigated by examining immunofluorescence. Proteins recognized by antibodies were identified by Western blot analysis. RESULTS: The prevalences of autoantibodies that bind to kidney tissues in cats were 41% and 13% by ELISA and immunofluorescence, respectively. Kidney-bound antibodies were observed at interstitial cells, apical border, and cytoplasm of proximal and distal tubules; the antibodies were bound to proteins with molecular weights of 40, 47, 38, and 20 kDa. There was no direct link between vaccination and anti-kidney antibodies, but positive antibodies to kidney tissues were significantly associated with the anti-FVRCP antibody. The odds ratio or association in finding the autoantibody in cats with the antibody to FVRCP was 2.8 times higher than that in cats without the antibody to FVRCP. CONCLUSIONS: These preliminary results demonstrate an association between anti-FVRCP and anti-cat kidney tissues. However, an increase in the risk of inducing kidney-bound antibodies by repeat vaccinations could not be shown directly. It will be interesting to expand the sample size and follow-up on whether these autoantibodies can lead to kidney function impairment.

Pilot study supporting the existence of novel lymphatic channels within the canine anterior uveal tract using Lyve-1 and CD31.

OBJECTIVE: To demonstrate the existence of lymphatics in the canine anterior uvea using lymphatic-specific markers Lyve-1, Prox-1, and podoplanin, the endothelial cell marker CD31, and basement membrane matrix marker collagen IV. DESIGN: Prospective Study. ANIMALS: Eight normal globes from animals euthanized for unrelated health problems. PROCEDURES: Sagittally cut serial sections of six normal canine eyes were immunofluorescence double-stained with Lyve-1 and CD31 and single-stained with colorimetric Prox-1 and collagen IV. Three serial sections from 2 additional eyes were cut in the coronal plane at the level of the ciliary body and immunofluorescence double-stained with Lyve-1 and CD31 to map lymphatic channel distribution. Lymphatics from normal canine lymph nodes were used for validation of podoplanin. RESULTS: Four of 6 of the sagitally sectioned eyes had Lyve-1-positive lymphatic-like structures that were distinct from CD31-positive blood vessels in the iris base and ciliary body. Both of the coronally sectioned globes had Lyve-1-positive lymphatic-like structures in the ciliary body. The location of these structures was evaluated and found to be diffusely present circumferentially around the ciliary body. CONCLUSION AND CLINICAL RELEVANCE: These results support the existence of lymphatic channels in the anterior uveal tract of the canine eye. This could indicate the presence of a novel uveolymphatic outflow pathway, which may play a role in aqueous humor outflow. Future studies are needed to confirm the existence and elucidate the role of this proposed uveolymphatic outflow pathway and potentially develop novel treatment options for managing glaucoma.

A rapid multiple immunofluorescence method for the simultaneous detection of CD20 and CD3 in canine and feline cytological samples.

CD20 and CD3 are considered reliable markers for B and T cells, respectively. This study aimed to develop a rapid multiple immunofluorescence (RMIF) method for the detection of CD20 and CD3 on a single cytology slide. Air-dried smears were prepared using samples collected from dogs (n=26) and cats (n=6). Immunosignal detection using the newly developed method required 60 min. Clear immunosignals for CD20 and CD3 were detected in 24 of 26 samples in dogs and in all 6 cats. As the RMIF (CD20/CD3) method can detect markers of both B and T cells simultaneously on a single cytology smear, it would be an efficient tool for the immunophenotyping of canine and feline lymphoma samples.

Characterization of neoplastic cells outlining the cystic space of invasive micropapillary carcinoma of the canine mammary gland.

BACKGROUND: Invasive micropapillary carcinoma (IMPC) is a rare malignant breast tumor and a variant form of invasive ductal carcinoma that is an aggressive neoplasm of the human breast and canine mammary gland. The importance of the tumor microenvironment in cancer development has gradually been recognized, but little is known about the cell types outlining the cystic space of canine IMPC. This study aimed to characterize the neoplastic cells outlining the cystic space of IMPC. RESULTS: Immunohistochemistry (IHC), immunofluorescence (IF), superresolution and transmission electron microscopy (TEM) were used to assess the cell types in the cystic areas of IMPCs. Cells expressing the mesenchymal markers alpha-smooth muscle actin (αSMA), Vimentin, and S100A4 outlined the cystic space of IMPC. Furthermore, loss of epithelial cell polarity in IMPC was shown by the localization of MUC1 at the stroma-facing surface. This protein modulates lumen formation and inhibits the cell-stroma interaction. Immunohistochemical and IF staining for the myoepithelial cell marker p63 were negative in IMPC samples. Furthermore, associated with peculiar morphology, such as thin cytoplasmic extensions outlining cystic spaces, was observed under TEM. These observations suggested cells with characteristics of myoepithelial-like cells. CONCLUSIONS: The cells outlining the cystic space of IMPC in the canine mammary gland were characterized using IHC, IF and TEM. The presence of cells expressing αSMA, Vimentin, and S100A4 in the IMPC stroma suggested a role for tumor-associated fibroblasts in the IMPC microenvironment. The reversal of cell polarity revealed by the limited basal localization of MUC1 may be an important factor contributing to the invasiveness of IMPC. For the first time, the cystic space of canine mammary gland IMPC was shown to be delimited by myoepithelial-like cells that had lost p63 expression. These findings may enhance our understanding of the cellular microenvironment of invasive tumors to improve cancer diagnosis and treatment.

Minimizing the risk of occupational Q fever exposure: A protocol for ensuring Coxiella burnetii-negative pregnant ewes are used for medical research.

Q fever is a worldwide zoonosis caused by Coxiella burnetii that can lead to abortion, endocarditis, and death in humans. Researchers utilizing parturient domestic ruminants, including sheep, have an increased risk of occupational exposure. This study evaluated the effectiveness of our screening protocol in eliminating C. burnetii-positive sheep from our facility. From August 2010 to May 2018, all ewes (N = 306) and select lambs (N = 272; ovis aries) were screened twice for C. burnetii utilizing a serum Phase I and Phase II antibody immunofluorescence assay (IFA). The first screen was performed by the vendor prior to breeding, and the second screen was performed on arrival to the research facility. Ewes that were positive on arrival screening were quarantined and retested using repeat IFA serology, enzyme-linked immunosorbent assay, buffy coat polymerase chain reaction (PCR), and amniotic fluid PCR. The overall individual seroprevalence of C. burnetii in the flocks tested by the vendor was 14.2%. Ewes with negative Phase I and Phase II IFA results were selected for transport to the research facility. Upon arrival to the facility, two (0.7%) ewes had positive Phase I IFA results. Repeat testing demonstrated seropositivity in one of these two ewes, though amniotic fluid PCR was negative in both. The repeat seropositive ewe was euthanized prior to use in a research protocol. No Q fever was reported among husbandry, laboratory or veterinary staff during the study period. Serologic testing for C. burnetii with IFA prior to transport and following arrival to a research facility limits potential exposure to research staff.

CD3-CD20-positive nodal lymphoma with cross-lineage rearrangement in a dog.

A 7-y-old mixed-breed male dog was presented with a history of generalized lymphadenopathy. Fine-needle aspirates of the enlarged peripheral lymph nodes were suggestive of lymphoma. Histologic examination of a retromandibular lymph node was suggestive of high-grade, medium large-cell lymphoma. Immunohistochemistry revealed concurrent expression of CD3 and CD20. The co-localization of the 2 antigens was confirmed by immunofluorescence. PCR for antigen receptor gene rearrangements (PARR) detected clonal rearrangements for both T-cell receptor gamma and B-cell receptor. The final diagnosis was CD3-CD20-positive anaplastic lymphoma with cross-lineage rearrangement.

Autoradiography and inmmunolabeling suggests that lizard blastema contains arginase-positive M2-like macrophages that may support tail regeneration.

BACKGROUND: The regenerating blastema of the tail in the lizard Podarcis muralis contains numerous macrophages among the prevalent mesenchymal cells. Some macrophages are phagocytic but others are devoid of phagosomes suggesting that they have other roles aside phagocytosis. METHODS: The presence of healing macrophages (M2-like) has been tested using autoradiographic, immunohistochemical and ultrastructural studies. RESULTS: Autoradiography shows an uptake of tritiated arginine in sparse cells of the blastema and in the regenerating epidermis. Bioinformatics analysis suggests that epitopes for arginase-1 and -2, recognized by the employed antibody, are present in lizards. Immunofluorescence shows sparse arginase immunopositive macrophages in the blastema and few macrophages also in the apical wound epidermis. The ultrastructural study shows that macrophages contain dense secretory granules, most likely inactive lysosomes, and small cytoplasmic pale vesicles. Some of the small vesicles are arginase-positive while immunolabeling is very diffuse in the macrophage cytoplasm. CONCLUSIONS: The presence of cells incorporating arginine and of arginase 1-positive cells suggests that M2-like macrophages are present among mesenchymal and epidermal cells of the regenerative tail blastema. M2-like macrophages may promote tail regeneration differently from the numerous pro-inflammatory macrophages previously detected in the scarring limb. The presence of M2-like macrophages in addition to hyaluronate, support the hypothesis that the regenerative blastema of the tail in lizards is an immuno-privileged organ where cell proliferation and growth occur without degenerating in a tumorigenic outgrowth.

Evaluation of a rapid immunochromatographic test kit to the gold standard fluorescent antibody test for diagnosis of rabies in animals in Bhutan.

BACKGROUND: Rabies kills approximately 59,000 people each year worldwide. Rapid and accurate diagnosis of rabies is important for instituting rapid containment measures and for advising the exposed people for postexposure treatment. The application of a rapid diagnostic tests in the field can greatly enhance disease surveillance and diagnostic activities, especially in resource poor settings. In this study, a total of 179 brain tissue samples collected from different rabies suspect animal species (113 dogs, 50 cattle, 10 cats, 3 goats, 2 horses, and 1 bear) were selected and tested using both rapid immunochromatographic kit and the reference standard fluorescent antibody test (FAT). We evaluated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of a rapid antigen detection test kit produced by BioNote, Inc. (Hwaseong-si, Korea) relative to a FAT for its fit-for-purpose for confirmation of clinical cases of rabies for early response and enhancing rabies surveillance. RESULTS: Among 179 samples examined in this study, there was a concordance in results by the rapid test and FAT in 115 positive samples and 54 negative samples. Test results were discordant in 10 samples which were positive by FAT, but negative (false negative) by rapid kit. The rapid test kit showed a sensitivity of 92% (95% CI: 85.9-95.6) and specificity of 100% (95% CI: 93.4-100) using FAT as the reference standard. The positive and negative predictive values were found to be 100% (95% CI:96.7-100) and 84.4% (95% CI: 73.6-91.3), respectively. Overall, there was 94.4% (95% CI: 90-96.9) test agreement between rapid test and FAT (Kappa value = 0.874) with a positive percent agreement and negative percent agreement of 92 and 100%, respectively. CONCLUSIONS: Our finding demonstrated that the rapid test kit (BioNote) can be used for rabies surveillance and confirming clinical case of rabies in animals for making rapid decisions particularly controlling rabies outbreaks in resource poor settings.

Antiviral Effects of Hydroxychloroquine and Type I Interferon on In Vitro Fatal Feline Coronavirus Infection.

Feline infectious peritonitis (FIP) is a viral disease with a high morbidity and mortality by the FIP virus (FIPV, virulent feline coronavirus). Several antiviral drugs for FIP have been identified, but many of these are expensive and not available in veterinary medicine. Hydroxychloroquine (HCQ) is a drug approved by several countries to treat malaria and immune-mediated diseases in humans, and its antiviral effects on other viral infections (e.g., SARS-CoV-2, dengue virus) have been confirmed. We investigated whether HCQ in association with interferon-ω (IFN-ω) is effective for FIPV in vitro. A total of 100 µM of HCQ significantly inhibited the replication of types I and II FIPV. Interestingly, the combination of 100 µM of HCQ and 104 U/mL of recombinant feline IFN-ω (rfIFN-ω, veterinary registered drug) increased its antiviral activity against type I FIPV infection. Our study suggested that HCQ and rfIFN-ω are applicable for treatment of FIP. Further clinical studies are needed to verify the combination of HCQ and rIFN-ω will be effective and safe treatment for cats with FIP.

Immunolocalization of corneous beta proteins of the Epidermal Differentiation Complex in the developing claw of the alligator.

Knowledge on the sharpness, mechanical and hydration resistance of the corneous material of claws requires information on its constituent proteins. The present immunohistochemical study has localized some of the main corneous beta proteins (CBPs, formerly termed beta-keratins) indicated to be present in alligator claws only by genomic data. Using specific antibodies we show the immunolocalization of representative claws CBPs of the Epidermal Differentiation Complex (Beta A1 group) during late stages of claw development in alligator. Intense but asymmetric proliferation, revealed by 5BrdU-immunolabeling, determines the formation of a curved dorsal part (unguis) and a linear ventral part (sub-unguis). The large beta-cells generated in the unguis and their packing into a solid corneous layer occur before thinner beta-cells appear in the sub-unguis. In the latter, CBPs are also immune-detected but with less intensity compared to the unguis, and corneocytes remain separated and desquamate. It is suggested that at the tip of the developing claw beta-corneocytes move downward into the initial part of the sub-unguis. This circular movement contributes to sharpen the claw as these cells fully cornify and are desquamated from the sub-unguis. Corneocytes of the unguis contain 10-16 kDa proline-serine-rich proteins that also possess high percentages of glycine, cysteine, tyrosine, valine and leucine. Cysteines likely give rise to numerous SS bonds in the constituent hard horny material, tyrosine contribute to packing proteins into a dense horny material while glycine, valine and leucine increase the hydrophobic property of claws in these water-adapted predators.

Approaches to overcome flow cytometry limitations in the analysis of cells from veterinary relevant species.

BACKGROUND: Flow cytometry is a powerful tool for the multiparameter analysis of leukocyte subsets on the single cell level. Recent advances have greatly increased the number of fluorochrome-labeled antibodies in flow cytometry. In particular, an increase in available fluorochromes with distinct excitation and emission spectra combined with novel multicolor flow cytometers with several lasers have enhanced the generation of multidimensional expression data for leukocytes and other cell types. However, these advances have mainly benefited the analysis of human or mouse cell samples given the lack of reagents for most animal species. The flow cytometric analysis of important veterinary, agricultural, wildlife, and other animal species is still hampered by several technical limitations, even though animal species other than the mouse can serve as more accurate models of specific human physiology and diseases. RESULTS: Here we present time-tested approaches that our laboratory regularly uses in the multiparameter flow cytometric analysis of ovine leukocytes. The discussed approaches will be applicable to the analysis of cells from most animal species and include direct modification of antibodies by covalent conjugation or Fc-directed labeling (Zenon™ technology), labeled secondary antibodies and other second step reagents, labeled receptor ligands, and antibodies with species cross-reactivity. CONCLUSIONS: Using refined technical approaches, the number of parameters analyzed by flow cytometry per cell sample can be greatly increased, enabling multidimensional analysis of rare samples and giving critical insight into veterinary and other less commonly analyzed species. By maximizing information from each cell sample, multicolor flow cytometry can reduce the required number of animals used in a study.

Comparison of immunofluorescence and chemiluminescence assays for measuring ACTH in equine plasma.

BACKGROUND: The chemiluminescence (CL) and immunofluorescence (IF) assays yield different results for basal adrenocorticotropin hormone concentrations [ACTH] in pony plasma. It is unclear whether this difference also occurs in basal samples from horses or samples from ponies following thyrotropin-releasing hormone (TRH) stimulation. OBJECTIVES: To compare the results of [ACTH] analysis by CL and IF methods in basal samples from horses and pony samples following TRH stimulation. STUDY DESIGN: Method comparison. METHODS: Plasma [ACTH] was measured concurrently using CL and IF methods in 12 ponies (basal and post-TRH stimulation) in November and basal samples from horses (n = 45; November and May). RESULTS: CL and IF methods yielded different results (P < .01). The median difference (CL-IF) (95% CI) for ponies was 5.9 (0.1-7.5) pg/mL at baseline and 227.9 (61-1001) pg/mL post TRH; and horses 1.9 (1.1-5.4) pg/mL in November and 9.4 (8.2-11.5) pg/mL in May, at baseline. Correlation was good in ponies at baseline (R = 0.80, P = .003) but not post-TRH, and good in horses in November and May (R = 0.68 and 0.71, P < .001). Bland-Altman analysis demonstrated moderate bias and wide 95% limits of agreement (95% LOA) in ponies at baseline (bias 5.5 pg/mL; 95% LOA -9.9 to 20.9 pg/mL) and horses in May (bias 10.6 pg/mL; 95% LOA -9 to 30.3 pg/mL) and very large bias and wide 95% LOA in ponies post-TRH (bias 477 pg/mL; 95% LOA -633 to 1587 pg/mL). Using CL cut-offs of >29 and >110 pg/mL, agreement was moderate (ƙ = 0.67) and very good (ƙ = 0.82) for binary classification of PPID in ponies at baseline and post-TRH; and good (ƙ = 0.73) for horses in November, but poor (ƙ = 0.40) in May. MAIN LIMITATIONS: Limited numbers of horses with [ACTH] above threshold values. CONCLUSIONS: The assays yielded different absolute values, particularly in post-TRH samples from ponies, suggesting TRH stimulates secretion of cross-reacting peptides other than ACTH. Agreement for binary classification for PPID was moderate to good, except in basal samples from horses in May.

Geese not susceptible to virulent subgroup J avian leukosis virus isolated from chickens.

To determine whether geese are susceptible to infection by avian leukosis virus (ALV), 702 serum samples from domestic and foreign goose breeds were screened for p27 antigen as well as being inoculated into DF-1 cell cultures to isolate ALV. Although 5.7% of samples were positive for p27 antigen, reactivity appeared to be non-specific because no ALV was detected in the corresponding DF-1 cultures. To further determine whether geese are susceptible to ALV-J isolated from chickens, ALV-J strain JS09GY7 was artificially inoculated into 10-day-old goose embryos, with one-day-old hatched goslings then screened for p27 antigen and the presence of ALV. In all cases, the results of both tests were negative. Liver tissues from the 1-day-old goslings were screened using a polymerase chain reaction-based assay, which failed to amplify ALV-J gene fragments from any of the samples. Further, no histopathological damage was observed in the liver tissues. ALV-J was further inoculated intraperitoneally into one-day-old goslings, with cloacal swabs samples and plasma samples then collected every 5 days for 30 days. All samples were again negative for the presence of p27 antigen and ALV, and liver tissues from the challenged geese showed no histopathological damage and were negative for the presence of ALV-J gene fragments. Furthermore, p27 antigen detection, PCR-based screening, and indirect immunofluorescence assays were all negative following the infection of goose embryo fibroblasts with ALV-J. Together, these results confirm that virulent chicken-derived ALV-J strains cannot infect geese, and that p27 antigen detection in goose serum is susceptible to non-specific interference.

Muscular Sarcocystosis in Sheep Associated With Lymphoplasmacytic Myositis and Expression of Major Histocompatibility Complex Class I and II.

Sarcocystosis is a protozoal disease affecting a wide range of animals. The aims of this study were to characterize the following in sheep: (1) the muscle pathology in Sarcocystis infection, (2) the inflammatory infiltrate and its relationship to severity of infection, and (3) immune markers expressed by parasitized muscle fibers and parasitic cysts. Skeletal muscle samples from 78 sheep slaughtered in southern Italy were snap frozen and analyzed by histopathology, immunohistochemistry, and immunofluorescence. Polymerase chain reaction (PCR) and sequencing were used for Sarcocystis species identification. All 40 muscle samples tested were PCR-positive for Sarcocystis tenella. Histologically, cysts were identified in 76/78 cases (97%), associated with an endomysial infiltrate of lymphocytes and plasma cells. The T cells were predominantly CD8+, with fewer CD4+ or CD79α+ cells. Eosinophils were absent. Notably, sarcolemmal immunopositivity for major histocompatibility complex (MHC) I and II was found in 76/78 cases (97%) and 75/78 cases (96%), respectively, both in samples with and in those without evident inflammatory infiltrate. The number of cysts was positively correlated with inflammation. In addition, MHC I was detected in 55/78 cyst walls (72%), and occasionally co-localized with the membrane-associated protein dystrophin. The findings suggest that muscle fibers respond to the presence of cysts by expression of MHC I and II. The possible role of MHC I and II in the inflammatory response and on the cyst wall is also discussed.

Serologic cross-reactivity between Sarcocystis neurona and Sarcocystis falcatula-like in experimentally infected Mongolian gerbils.

Sarcocystis neurona is the major cause of the equine protozoal myeloencephalitis (EPM) in the Americas and has opossums of the genus Didelphis as definitive hosts. Most isolates of Sarcocystis sp. shed by opossums in Brazil differ genetically from the known species of Sarcocystis. These Brazilian isolates behave similarly as Sarcocystis falcatula, which causes sarcocystosis in birds, and for this reason, have been classified as Sarcocystis falcatula-like. Genes coding for the immunodominant surface antigens SAG2, SAG3 and SAG4 of S. falcatula-like are similar to those from S. neurona. It is unknown the Sarcocystis species that causes EPM in Brazil, as S. neurona has never been genetically confirmed in Brazilian horses. All cases associated with EPM in Brazil were diagnosed by immunological tests, which are not specific for S. neurona infection. It is possible that S. falcatula-like may infect horses in Brazil. The aims of the current study were to test the susceptibility of gerbils (Meriones unguiculatus) to experimental infections with S. neurona and S. falcatula-like, and to investigate potential serologic cross-reactivity to these parasites by immunofluorescent antibody test (IFAT) and Western blot (WB). A total of 27 gerbils, distributed in five experimental groups (G1-G5), were employed in this work (G1: 4 negative controls; G2: 6 infected with S. neurona merozoites, G3: 6 infected with S. falcatula-like merozoites; G4 and G5 (5 and 6, respectively, infected with different doses of sporocysts). None of the 17 animals that seroconverted for the parasites in IFAT presented any visualized organism or Sarcocystis DNA in the examined tissues. No serologic cross-reactivity was observed using IFAT. However, sera from animals infected with S. falcatula-like and S. neurona presented the same pattern of antigenic recognition when S. neurona merozoites were used as antigen in WB, including reactivity to proteins of 30 and 16 kDa, regarded as specific markers for S. neurona-infected animals. Gerbils did not sustain infection by these parasites, although produced antibodies after inoculation. These results are suggestive that other animal species that are exposed to S. falcatula-like, including horses, may present serologic cross-reactivity to S. neurona in WB. IFAT was demonstrated to be more specific that WB for the detection of antibodies to S. falcatula-like and S. neurona in the experimental conditions of this study.

Molecular and serological survey of carnivore pathogens in free-roaming domestic cats of rural communities in southern Chile.

Owned, free-roaming domestic cats are abundant in the Chilean countryside, having high probability of contact with wildlife and potentially participating as reservoirs of zoonotic pathogens. In the present study, 131 cats from two remote study areas (Valdivia and Chiloe Island) in southern Chile were analyzed for infection/exposure to eight pathogens. Serum samples from 112 cats were tested for antigens against feline leukemia virus (FeLV antigen-ELISA) and antibodies against feline immunodeficiency virus (FIV-ELISA) and canine distemper virus (CDV-serum neutralization), yielded occurrence of 8.9, 1.7 and 0.8% respectively. The presence of DNA of five vector-borne pathogens, piroplasmids, Ehrlichia spp., Anaplasma spp., Rickettsia spp. and Bartonella spp. was investigated in thirty cats. Overall observed occurrence was 6.6% (2/30) for both Anaplasma platys, and B. henselae, and 3.3% (1/30) for both Bartonella sp. and Theileria equi. Observed occurrence for all vector-borne pathogens in Valdivia area was significantly higher than in Chiloe Island (5/15 vs 0/15; P=0.04). Our results represent the first description of exposure to CDV and DNA detection of T. equi and A. platys in domestic cats in Chile. The results highlight the importance of performing pathogen screening in owned, free-roaming rural cats to evaluate their potential role as reservoirs of infection and vectors for disease transmission to wildlife.

Eimeria tenella induces the release of chicken heterophil extracellular traps.

Avian coccidiosis makes a great threat and economic loss to the poultry industry, and fully understanding the innate immune response of chicken against E. tenella infection will play a significant role in avian coccidiosis prevention and treatment. Extracellular traps have been reported as a novel defense mechanism of host against pathogens infection. However, the interaction between chicken heterophil extracellular traps and E. tenella has remained not well known. Thus, this study aims to investigate the effects of E. tenella on chicken heterophil extracellular traps (ETs), and try to clarify the regulatory mechanisms in this process. E. tenella-triggered chicken heterophil ETs structures were analyzed by using scanning electron microscopy (SEM) and scanning confocal microscope. Inhibitors and Pico Green® were used to quantify E. tenella - triggered chicken heterophil ETs release. The results showed that E. tenella sporozoites significantly induced chicken heterophil ETs-like structures release, and histone and elastin co-existed with DNA in these structures of chicken heterophil ETs. Furthermore, it was also demonstrated that NADPH, p38 or Rac1 signaling pathways participated in E. tenella sporozoites-induced chicken heterophil ETs release, but more key molecules or signaling pathways involved in this process still needed to be further investigated. Taken together, this study reports that E. tenella sporozoites could induce chicken heterophil ETs formation via NADPH, p38 and Rac1 signaling pathways, which further suggests the critical role of heterophil ETs in the process of chicken against E. tenella infection.