Surra-affected dromedary camels show reduced numbers of blood B-cells and in vitro evidence of Trypanosoma-induced B cell death.

Trypanosomosis due to Trypanosoma evansi (surra) is one of the most important diseases with a significant impact on camel health and production. Trypanosoma-induced immunosuppression mechanisms, which are key factors of disease pathogenesis, have been characterized in several animal species. The present study investigated, therefore, the impact of trypanosomosis on the immunophenotype of blood leukocytes in camels. For this, the relative and absolute values of blood leukocyte populations, their expression pattern of cell surface molecules, and the numbers of the main lymphocyte subsets were compared between healthy camels and camels with clinical symptoms of chronic surra and serological evidence of exposure to Trypanosoma infection. Leukocytes were separated from the blood of healthy and diseased camels, labeled with fluorochrome-conjugated antibodies, and analyzed by flow cytometry. Compared to healthy camels, the leukogram of diseased camels was characterized by a slightly increased leukocyte count with moderate neutrophilia and monocytosis indicating a chronic inflammatory pattern that may reflect tissue injury due to the long-lasting inflammation. In addition, the analysis of lymphocyte subsets revealed a lower number and percentage of B cells in diseased than healthy camels. In vitro incubation of camel mononuclear cells with fluorochrome-labeled T. evansi revealed a higher capacity of camel B cells than T cells to bind the parasite in vitro. Furthermore, cell viability analysis of camel PBMC incubated in vitro with T. evansi whole parasites but not the purified antigens resulted in Trypanosoma-induced apoptosis and necrosis of camel B cells. Here we demonstrate an association between trypanosomosis in camels and reduced numbers of blood B cells. In vitro analysis supports a high potential of T. evansi to bind to camel B cells and induce their elimination by apoptosis and necrosis.

Flow cytometric analysis of immune cell populations in the bronchial and mesenteric lymph nodes of the dromedary camel.

Dromedary camel is an important livestock species with special economic value in arid and semi-arid regions of the world. Given the limited data on detailed immune cell composition and cell marker expression in the dromedary camel lymph node tissue, the present study was undertaken to investigate the immune cell composition of bronchial and mesenteric lymph nodes from healthy dromedary camels using flow cytometry. In this study, we applied flow cytometry and multicolor immuno-fluorescence to phenotype the main populations of immune cells in the bronchial and mesenteric camel lymph nodes and compared them with separated peripheral blood mononuclear cells and granulocytes. We used antibodies to detect several cell surface molecules associated with camel T cells (CD4, WC1), B cells (MHCII, BAQ44A), monocytes/macrophages (CD172a, CD14, CD163), in addition to the pan-leukocyte marker CD45 and the cell adhesion molecules CD44 and CD18. Compared to blood mononuclear cells, camel lymph node cells contained a higher percentage of lymphoid cells with only a minor fraction of myeloid cells. In addition, the lower expression of CD44 and CD18 on lymph node lymphocytes compared to lymphocytes from peripheral blood indicates higher frequency of naïve lymphocytes in the lymph nodes. The frequency of CD4+ T cells, B cells and γδ T cells within camel lymph node lymphocytes compared to blood indicates a similar tissue distribution pattern of lymphocyte subsets in camel and bovine and supports previous reports on the similarity between the camel immune system and the immune system of other ruminants. Lymph node neutrophils were identified as CD45++ CD172a++, CD14+, MHCIIlow, BAQ44A+, CD44++, CD18++ cells. In conclusion, the present study is describing the employment of flow cytometric single-cell analysis and immunostaining for the analysis of the immune cell composition in the camel lymph node.

Detection of Avian Orthoavulavirus-1 genotypes VI.2.1 and VII.1.1 with neuro-viscerotropic tropism in some backyard pigeons (Columbidae) in Eastern Saudi Arabia.

Introduction: Avian orthoavulavirus-1 (AOAV1) has a wide host range, including domestic and wild birds. The present study aimed to identify the currently circulating AOAV1 strains from some outbreaks in some backyard pigeons in the eastern region of Saudi Arabia (ERSA). Methods: Tracheal/cloacal swabs and tissue specimens were collected from eight backyards in Al-Ahsa, ERSA, between January 2021 and March 2023. Samples were tested for the presence of AOAV1 using commercial real-time RT-PCR. Part of the fusion gene was also amplified by gel-based RT-PCR, and the obtained amplicons were sequenced. Results and discussion: AOAV1 was detected in samples from the eight flocks. The retrieved sequences from samples of 6/8 pigeon backyards are reported. Phylogenetic analysis based on the obtained sequences from these backyard pigeons showed the segregation of the obtained sequences in AOAV1 genotypes VI.2.1 and VII.1.1. Clinically, nervous manifestations were dominant in pigeons infected with both genotypes. Respiratory manifestations and significantly higher overall mortality rate were induced by genotype VI.2.1. The deduced amino acid sequences of the fusion protein cleavage site (FPCS) showed that all the detected isolates belong to velogenic strains. Differences in clinical profiles induced by the natural infection of pigeons with AOAV1 genotypes VI.2.1 and VII.1.1 were reported. The present findings highlight the potential roles of some backyard pigeons in the long-distance spread and cross-species transmission of the reported AOAVI genotypes. Further research is required to perform biotyping and pathotyping of the reported strains.

Avian encephalomyelitis virus in backyard chickens.

Background and Aim: Avian viral diseases usually cause high economic losses because of high morbidity and mortality and poor growth. The rearing of chickens in backyards could have an important role in the spread of certain diseases, particularly those of viral origin. Infected birds might be prone to many viral infections for several reasons, including a lack of vaccination programs, the mixing of different bird species in the same location, and the close interactions of these birds with wild and migratory birds carrying various pathogens. This study aimed to conduct serological surveillance of avian encephalomyelitis virus (AEV) in some backyard chickens in the eastern region of Saudi Arabia. Materials and Methods: Serum samples (n = 368) were collected from domestic chickens reared in 10 backyards in the Eastern Province of Saudi Arabia. None of the domestic birds in these 10 backyards were vaccinated against the virus. In addition, 78 serum samples were collected from free-ranging birds belonging to Columbidae, such as pigeons and doves, in common areas near the domestic backyards. We tested these sera for specific antibodies against AEV. Results: Our results revealed seroconversion to AEV among the examined chickens (14.6%). None of the tested pigeons and doves displayed seroconversion to AEV. Conclusion: Seroconversion of these non-vaccinated birds against AEV was suggestive of a recent natural infection by this virus. Further studies with a large number of birds are required to molecularly characterize the circulating strains of this virus in this area.

Evidence of the circulation of avian metapneumovirus in domestic backyard chickens in Eastern Saudi Arabia in 2019.

Background and Aim: Avian metapneumovirus (aMPV) is a recently discovered respiratory virus in chickens. Avian metapneumovirus has been linked to respiratory syndromes, reproductive failure in affected chickens and turkeys, swollen head syndrome in chickens, and rhinotracheitis in turkeys. Wild birds are considered potential reservoirs of aMPV, particularly aMPV-C. However, little is known about the prevalence of aMPV in Saudi Arabia. Considering the relevance of backyard chickens in the transmission and sustainability of certain avian viral diseases, this study aimed to assess aMPV exposure in backyard chickens and wild birds circulating near selected locations. Materials and Methods: We collected 368 serum samples from unvaccinated backyard chickens in ten locations in Eastern Saudi Arabia. Furthermore, we collected 78 serum samples from species of free-ranging birds belonging to the Columbidae family, such as pigeons and doves, captured from the same areas. Using commercial enzyme-linked immunosorbent assay kits, we tested the sera of domestic backyard chickens and wild birds for antibodies against aMPV. Results: Our results showed that 74/368 birds were positive for aMPV-related antibodies. Conversely, none of the tested wild birds seroconverted to aMPV. Conclusion: The antibody titers detected in the backyard chickens suggested recent exposure to aMPV. Considering these results, further large-scale serological and molecular studies are needed to evaluate the prevalence of aMPV in these birds and characterize the circulating strains of aMPV in this region.

Impact of Selected Bacterial and Viral Toll-like Receptor Agonists on the Phenotype and Function of Camel Blood Neutrophils.

Innate recognition of pathogens depends on the interaction between microbial structures known as pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) in host cells. Toll-like receptors (TLR) are among the most important PRRs being expressed on and in a wide range of immune cell types. Studies on the interaction mechanisms between different pathogen species and the immune system of the dromedary camel are still scarce. The present study aimed to investigate the immunomodulatory effect of synthetic bacterial and viral TLR ligands on some phenotypic properties and selected functions of neutrophils purified from dromedary camel blood. Neutrophils were separated from camel blood (n = five animals) and were stimulated in vitro with the TLR ligands LPS, Pam3CSK4, R848 (Resiquimod), and Poly IC or were left without stimulation. Stimulation with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was used as a positive control stimulation. Shape change, phagocytosis activity, ROS production, the expression of cell surface markers, and cell vitality were compared between stimulated and non-stimulated cells. With exception of the TLR3 agonist Poly IC, all TLR ligands used showed the potential to stimulate camel neutrophils resulting in increased cell size and the upregulation of CD18 and CD14 on their surface. Similarly, the phagocytosis activity of camel neutrophils was significantly improved after priming with all TLR ligands, except Poly IC, which, in contrast, resulted in a reduced percentage of phagocytosis-positive cells. In contrast to stimulation with PMA, which induced a significant ROS production in camel neutrophils, none of the TLR ligands used stimulated ROS generation in neutrophils. Only stimulation with Pam3CSK4 increased the expression of MHCII molecules on camel neutrophils, resulting in an expanded MHCIIhigh fraction within camel neutrophils. Our study indicates selective immunomodulating effects of TLR agonists on purified camel neutrophils without affecting their vitality.

Immunomodulatory Effects of Bacterial Toll-like Receptor Ligands on the Phenotype and Function of Milk Immune Cells in Dromedary Camel.

(1) Toll-like receptors (TLR) are a family of pattern recognition receptors that sense distinct molecular patterns of microbial origin. Although the immune cell composition of camel milk has been recently described, host-pathogen interaction studies in the camel mammary gland are still scarce. The present study aimed to use a whole milk stimulation assay for investigating the modulatory effect of selected Toll-like receptor (TLR) ligands on the phenotype and function of milk immune cells. (2) Methods-camel milk samples (n = 7) were stimulated in vitro with the TLR4 ligand LPS or the TLR2/1 ligand Pam3CSK4, and separated milk cells were evaluated for stimulation-induced shape change, the expression of cell surface markers, phagocytosis, apoptosis, ROS production, and NETosis. Stimulation with PMA was used as a control stimulation. (3) Results-all stimulants induced shape change in milk cells, change in the expression of several cell markers, and increased cell apoptosis and NETosis. In addition, stimulation with Pam3CSK4 and PMA was associated with enhanced ROS production, while only PMA stimulation resulted in enhanced bacterial phagocytosis by milk immune cells. (4) Conclusions-our data indicates selective modulating effects of the TLR ligands LPS and Pam3CSK4 on camel milk phagocytes. These results may have implications for the use of synthetic TLR agonists as immunomodulatory adjuvants of the immune response to intra-mammary vaccines against mastitis pathogens.

A Flow Cytometry Study of the Binding and Stimulation Potential of Inactivated Trypanosoma evansi toward Dromedary Camel Leukocytes.

Surra, a wasting disease caused by Trypanosoma evansi, is one of the major animal health burdens in camel-rearing countries, imposing significant economic losses due to reduced fertility and high mortality rates. The present study used inactivated T. evansi (from the Card Agglutination Test for Trypanosomes/Trypanosoma evansi; CATT/T. evansi) and flow cytometry to investigate their binding and activation potential toward camel leukocyte subsets. Labeling T. evansi with propidium iodide (PI) enabled their flow cytometric enumeration and identification with forward scatter (FSC; indicative for cell size) and side scatter (SSC; indicative for cell internal complexity) characteristics that are comparable with values reported for Trypanosoma cruzi. The incubation of PI-labeled non-opsonized T. evansi with camel leukocyte populations revealed that camel monocytes have the highest potential to bind T. evansi, followed by granulocytes and lymphocytes. The identification of pattern recognition receptors (PRRs) on camel immune cells and the pathogen-associated molecular patterns (PAMPs) in T. evansi that are responsible for this different binding capacity requires further studies. Stimulation of camel neutrophils with Trypanosoma evansi induced shape change, reactive oxygen species (ROS) production, and neutrophil extracellular traps (NET)-formation. To ensure that T. evansi, in the parasite concentration used in this study, is not apoptotic or necrotic to camel leukocytes, we evaluated cell apoptosis and necrosis after stimulation with T. evansi. The results revealed no impact of T. evansi stimulation for 2 h on the cell viability of camel leukocytes. Subsequent work may focus on the diagnostic employment of labeled T. evansi and flow cytometry for the detection of anti-Trypanosoma antibodies in camel serum. In addition, more efforts should be deployed to investigate the host-pathogen interaction mechanisms and the escape mechanisms of T. evansi in camels. To complete these data, further studies using the living or freshly killed parasites could also be implemented in camels and/or horses.

Risk-associated factors associated with the bovine viral diarrhea virus in dromedary camels, sheep, and goats in abattoir surveillance and semi-closed herd system.

Background and Aim: Bovine viral diarrhea virus (BVDV) is one of the most important viral pathogens causing high economic losses in cattle of all ages. Despite the active vaccination campaigns against BVDV, many outbreaks are still detected in various populations of cattle worldwide. Other species of animals such as dromedary camels, sheep, and goats may harbor BVDV infection and cause variable clinical syndromes. Thus, they may act as a source of infection to the cattle population around them. However, little is still known about the roles of these animals in the viral transmission and sustainability of BVDV in the environment. This study aimed to explore if the dromedary camels, sheep, and goats may seroconvert against BVDV and to study some associated risk factors for BVDV in these species of animals. Materials and Methods: We tested 1012 serum samples from dromedary camels, 84 from goats, and 21 from sheep for BVDV antibodies using commercial enzyme-linked immunosorbent assay (ELISA) kits. Meanwhile, we selected 211 serum samples from dromedary camels to be tested for the BVDV antigen using the commercial ELISA kits. Results: Our results show that 49/1117 serum samples were positive for the BVDV antibodies in dromedary camels (46/1012), goats (3/84), and none of the tested sheep samples were positive. However, none of the collected serum samples tested positive for the BVDV antigen. Conclusion: Seroconversion of some dromedary camels, sheep, and goats to the BVDV with no history of vaccination against BVDV strongly suggests the potential roles of these species of animals in the virus transmission cycle. The main limitations of the current study are (1) the lack of samples from other species of animals that lived close by these animals, particularly cattle. (2) lack of follow-up samples from the same animal over a long period. We believe the long-term longitudinal study of BVDV in various species of animals, particularly dromedary camels, goats, and sheep, is one of our future research directions. This will provide more information about the dynamics of BVDV antibodies in these species of animals.