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.

A longitudinal study of bovine viral diarrhea virus in a semi-closed management dairy cattle herd, 2020-2022.

Introduction: Bovine viral diarrhea virus (BVDV) brings great economic loss to the cattle industry worldwide. Developing a control/prevention strategy requires the prior assessment of certain epidemiological parameters. To determine the BVD incidence rate and associated risk factors, a dairy cattle herd in the eastern region of Saudi Arabia was monitored between 2020 and 2022. Methods: Nasal swabs (n = 190), rectal swabs (n = 190), and sera (n = 190) were collected from 79 cows in this herd. Collected sera and swabs were tested using the commercially available ELISAs for the BVDV antibodies and antigens, respectively. Collected sera were also tested for the presence of BVDV nucleic acids using commercial real-time RT-PCR kits. Results and discussion: Our data show BVDV seroprevalence (18.8%, 15%, and 8.2%) in the tested animals in 2020-2022, respectively. None of the collected nasal swabs, rectal swabs, or sera tested positive for the BVDV antigen, whereas 10.1%, 10%, and 18.1% of the tested sera were positive for BVDV nucleic acid in 2020-2022, respectively. The incidence rate was estimated at 0.02446 new cases/year despite the detection of BVDV in seronegative animals on single or two occasions at ≥6-month intervals. Young calves and bulls remained apparently unexposed to BVDV despite their presence with BVDV-infected females, with no significant physical separation. Both seropositivity and nucleic acid detectability showed significant positive and negative correlations, respectively, with reproductive performance. Collectively, the present study provides useful clues about the transmissibility of BVDV in the presence of possibly persistently infected animals. To the best of our knowledge, this is the first longitudinal study of BVDV in the Eastern Region of Saudi Arabia. Further detailed characterization of the circulating BVDVs is encouraged.

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.

Candidate genes associated with heat stress and breeding strategies to relieve its effects in dairy cattle: a deeper insight into the genetic architecture and immune response to heat stress.

The need for food products of animal origin is increasing worldwide. Satisfying these needs in a way that has minimal impact on the environment requires cutting-edge technologies and techniques to enhance the genetic quality of cattle. Heat stress (HS), in particular, is affecting dairy cattle with increasing frequency and severity. As future climatic challenges become more evident, identifying dairy cows that are more tolerant to HS will be important for breeding dairy herds that are better adapted to future environmental conditions and for supporting the sustainability of dairy farming. While research into the genetics of HS in the context of the effect of global warming on dairy cattle is gaining momentum, the specific genomic regions involved in heat tolerance are still not well documented. Advances in omics information, QTL mapping, transcriptome profiling and genome-wide association studies (GWAS) have identified genomic regions and variants associated with tolerance to HS. Such studies could provide deeper insights into the genetic basis for response to HS and make an important contribution to future breeding for heat tolerance, which will help to offset the adverse effects of HS in dairy cattle. Overall, there is a great interest in identifying candidate genes and the proportion of genetic variation associated with heat tolerance in dairy cattle, and this area of research is currently very active worldwide. This review provides comprehensive information pertaining to some of the notable recent studies on the genetic architecture of HS in dairy cattle, with particular emphasis on the identified candidate genes associated with heat tolerance in dairy cattle. Since effective breeding programs require optimal knowledge of the impaired immunity and associated health complications caused by HS, the underlying mechanisms by which HS modulates the immune response and renders animals susceptible to various health disorders are explained. In addition, future breeding strategies to relieve HS in dairy cattle and improve their welfare while maintaining milk production are discussed.

Evaluation of Hematological Profiles and Monocyte Subpopulations in Water Buffalo Calves after Immunization with Two Different IBR Marker Vaccines and Subsequent Infection with Bubaline alphaherpesvirus-1.

Bubaline alphaherpesvirus-1 (BuAHV-1) and Bovine alphaherpesvirus-1 (BoAHV-1) are respiratory viruses that can cause an infection known as "Infectious Bovine Rhinotracheitis" (IBR) in both water buffalo and bovine species. As the main disease control strategy, vaccination can protect animals from clinical disease through the development of specific humoral and cell-mediated immune responses. In the present study, the time-related circulatory kinetics of hematological profile and bubaline monocyte subsets have been investigated in vaccinated buffalo calves after challenge infections with BuAHV-1. Thirteen buffalo calves were selected and grouped into the VAX-1 group, which received an IBR-live-attenuated gE-/tk-deleted marker vaccine; the VAX-2 group, which received an IBR-inactivated gE-deleted marker vaccine; the CNT group, which remained an unvaccinated control. Fifty-five days after the first vaccination, the animals were infected with 5 × 105.00 TCID50/mL of wild-type BuAHV-1 strain via the intranasal route. Whole blood samples were collected at 0, 2, 4, 7, 10, 15, 30, and 63 days post-challenge (PCDs) for the analysis of hematological profiles and the enumeration of monocyte subsets via flow cytometry. The analysis of leukocyte compositions revealed that neutrophils were the main leukocyte population, with a relative increase during the acute infection. On the other hand, a general decrease in the proportion of lymphocytes was observed early in the post-infection, both for the VAX-1 and VAX-2 groups, while in the CNT group, the decrease was observed later at +30 and +63 PCDs. An overall infection-induced increase in blood total monocytes was observed in all groups. The rise was especially marked in the animals vaccinated with an IBR-live-attenuated gE-/tK-deleted marker vaccine (VAX-1 group). A multicolor flow cytometry panel was used to identify the bubaline monocyte subpopulations (classical = cM; intermediate = intM; and non-classical = ncM) and to investigate their variations during BuAHV-1 infection. Our results showed an early increase in cMs followed by a second wave of intMs. This increase was observed mainly after stimulation with live-attenuated viruses in the VAX-1 group compared with the animals vaccinated with the inactivated vaccine or the non-vaccinated animal group. In summary, the present study characterized, for the first time, the hematological profile and distribution of blood monocyte subsets in vaccinated and non-vaccinated water buffalo in response to experimental infection with BuAHV-1. Although not experimentally proven, our results support the hypothesis of a linear developmental relationship between monocyte subsets.

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.

Immunoinformatic prediction of the pathogenicity of bovine viral diarrhea virus genotypes: implications for viral virulence determinants, designing novel diagnostic assays and vaccines development.

Introduction: Bovine viral diarrhea virus (BVDV) significantly impacts the bovine industries, both dairy and beef sectors. BVDV can infect various domestic and wild animals, most notably cattle. The dynamic variations among BVDV serotypes due to the continuous genetic diversity, especially in BVDV1 (BVDV1), reduce the effectiveness of the currently available vaccines and reduce the specificity/sensitivity of the diagnostic assays. The development of novel, safe, and effective vaccines against BVDV requires deep knowledge of the antigenicity and virulence of the virus. Previous studies on the antigenicity and the virulence of BVDV serotypes have been mainly focused on one or a few BVDV proteins. While however, little is known about the orchestration of all BVDV in the context of viral virulence and immunogenicity. The main aim of the current study was to do a comparative computational evaluation of the immunogenicity, and virulence for all the encoded proteins of both BVDV1 and BVDV2 and their sub-genotypes. Methods: To achieve this goal, 11,737 protein sequences were retrieved from Virus Pathogen Resource. The analysis involved a total of 4,583 sequences after the removal of short sequences and those with unknown collection time. We used the MP3 tool to map the pathogenic proteins across different BVDV strains. The potential protective and the epitope motifs were predicted using the VaxiJen and EMBOSS antigen tools, respectively. Results and discussion: The virulence prediction revealed that the NS4B proteins of both BVDV1 and BVDV2 likely have essential roles in BVDV virulence. Similarly, both the capsid (C) and the NS4-A proteins of BVDV1 and the Npro and P7 proteins of BVDV2 are likely important virulent factors. There was a clear trend of increasing predicted virulence with the progression of time in the case of BVDV1 proteins, but that was not the case for the BVDV2 proteins. Most of the proteins of the two BVDV serotypes possess antigens predicted immunogens except Npro, P7, and NS4B. However, the predicted antigenicity of the BVDV1 was significantly higher than that of BVDV2. Meanwhile, the predicted immunogenicity of the immunodominant-E2 protein has been decreasing over time. Based on our predicted antigenicity and pathogenicity studies of the two BVDV serotypes, the sub-genotypes (1a, 1f, 1k, 2a, and 2b) may represent ideal candidates for the development of future vaccines against BVDV infection in cattle. In summary, we identified some common differences between the two BVDV genotypes (BVDV1 and BVDV2) and their sub-genotypes regarding their protein antigenicity and pathogenicity. The data presented here will increase our understanding of the molecular pathogenesis of BVDV infection in cattle. It will also pave the way for developing some novel diagnostic assays and novel vaccines against BVDV in the near future.

Flow Cytometric Identification and Enumeration of Monocyte Subsets in Bovine and Water Buffalo Peripheral Blood.

Monocytes are innate immune system key players with pivotal roles during infection and inflammation. They migrate into tissues and differentiate into myeloid effect cells (macrophages, dendritic cells) which orchestrate inflammatory processes and are interfaces between the innate and adaptive immune responses. Their clinical relevance to health and disease of cattle (Bos taurus) and water buffalo (Bubalus bubalis), two of the most important livestock species, has been highlighted in physiologic (pregnancy) and pathologic (mastitis, metritis, and viral infections) conditions. The existence of three different monocyte subsets in cattle was established by flow cytometry (FC), as follows: classical (cM; CD14++ CD16-/low ), intermediate (intM; CD14++/+ CD16+ ), and non-classical (ncM; CD14-/low CD16++ ) monocytes. FC applications for studying the immune system of cattle and water buffalo still have significant limitations. In this article, we describe some practical approaches to overcome these limitations and, in particular, allow the identification and enumeration of cM, intM, and ncM subpopulations in cattle and buffalo peripheral blood. Indeed, we propose the new procedure lyse/wash/no-centrifugation (L/W/NC) that can be combined with the FC absolute counting procedures and can overcome specific issues of the lyse/no-wash protocols (L/NW). Finally, for the first time, we demonstrated the existence of cM, intM, and ncM monocyte subsets also in the water buffalo, showing some interesting differences with cattle, such as the bubaline cM are mainly CD14+/++ /CD16+ . These subtle differences may influence inflammatory disease regulation in, for example, mastitis and metritis. The upregulation of CD16 expression on cM may reveal different monocyte priming, leading to different functional features of macrophages/dendritic cells in tissues after infection. © 2023 Wiley Periodicals LLC. Basic Protocol: Absolute count of cM, intM, and ncM without compensation Alternate Protocol: Absolute count of cM, intM, and ncM for single laser platform Support Protocol 1: In-house monoclonal antibody labeling using a Pacific Blue™ kit Support Protocol 2: In-house monoclonal antibody labeling using an Alexa Fluor® 647 kit Support Protocol 3: Titration of fluorochrome-conjugated antibodies.

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.

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.

Cytological and microbiological evaluation of conjunctiva in camels with and without conjunctivitis.

OBJECTIVE: To describe the cytological analysis of conjunctiva from normal camels and camels with bacterial conjunctivitis. ANIMALS STUDIED: This study was conducted on 7 normal camels and 15 camels affected with conjunctivitis. The affected camels had a history of conjunctivitis with signs including chemosis, blepharospasm, frequent blinking, and mild-to-moderate serous, mucoid, or purulent ocular discharge. PROCEDURES: Bacterial swabs were collected from the inferior conjunctival sac of the affected eye without topical anesthetics. Conjunctival smears were obtained from the conjunctival surface for cytological analysis. RESULTS: The cellular analysis of ocular smears revealed a higher percentage of basal cells, neutrophils, eosinophils, and macrophages in camels with conjunctivitis compared with normal camels. In contrast to this, smears from normal camels showed an increased percentage of superficial epithelial cells compared with affected camels. The microbiological assessment of conjunctival swabs collected from affected animals identified a bacterial growth of Staphylococcus aureus., Bacillus sp., Streptococcus sp., Enterococcus faecium., Staphylococcus sp., Corynebacterium sp., Coryne pseudotuberculosis., Saprophytica, Enterobacter cloacae, Escherichia coli, Proteus mirabilis, Proteus vulagaris, and Pseudomonas aeruginosa. CONCLUSIONS: It was observed that bacterial conjunctivitis in camels was associated with increased percentages of basal epithelial cells, neutrophils, eosinophils, and macrophages compared with normal camels, while normal camels showed an increased percentage of superficial epithelial cells compared with affected camels.

Hyperthermia-induced changes in leukocyte survival and phagocytosis: a comparative study in bovine and buffalo leukocytes.

Heat stress negatively affects health, welfare, and livestock productivity by impairing immune function, increasing disease incidence. In recent years, there has been increasing interest in understanding the immune system of water buffalo due to the growing economic impact of this species for the high quality and nutritional value of buffalo milk. While there are common responses across bovine and buffalo species, there are also some species-specific variations in the physiological responses to heat stress, mainly attributed to differences in metabolism and heat dissipation efficiency. At cellular level, the exposure to thermal stress induces several anomalies in cell functions. However, there is limited knowledge about the differential response of bovine and buffalo leucocytes to early and late exposure to different degrees of thermal exposure. The aim of this study was to compare the in vitro effect of hyperthermia on apoptosis and phagocytosis in leukocytes from bovine and buffalo species. For this, whole blood samples of six bovines and nine buffaloes were incubated at 39°C (mimicking normothermia condition) or 41°C (mimicking heat stress condition) for 1, 2, and 4 h. Two flow cytometric assays were then performed to evaluate apoptosis and determine functional capacity of phagocytic cells (neutrophils and monocytes). The results showed that the viability of bovine and buffalo leukocytes was differently affected by temperature and time of in vitro exposure. A higher percentage of apoptotic leukocytes was observed in bovines than in buffaloes at 39°C (3.19 vs. 1.51, p < 0.05) and 41°C (4.01 vs. 1.69, p < 0.05) and for all incubation time points (p < 0.05). In contrast, no difference was observed in the fraction of necrotic leukocytes between the two species. In both species, lymphocytes showed the highest sensitivity to hyperthermia, showing an increased apoptosis rates along with increased incubation time. In bovine, apoptotic lymphocytes increased from 5.79 to 12.7% at 39°C (p < 0.05), in buffalo, this population increased from 1.50 to 3.57% at 39°C and from 2.90 to 4.99% at 41°C (p < 0.05). Although no significant differences were found between the two species regarding the percentage of phagocytic neutrophils, lower phagocytosis capacity values (MFI, mean fluorescence intensity) were found in bovines compared with buffaloes at 41°C (27960.72 vs. 53676.45, p > 0.05). However, for monocytes, the differences between species were significant for both phagocytosis activity and capacity with lower percentages of bovine phagocytic monocytes after 2 h at 39°C and after 1 h at 41°C. The bovine monocytes showed lower MFI values for all temperature and time variations than buffaloes (37538.91 vs. 90445.47 at 39°C and 33752.91 vs. 70278.79 at 41°C, p < 0.05). In conclusion, the current study represents the first report on the comparative analysis of the effect of in vitro heat stress on bovine and buffalo leukocyte populations, highlighting that the leukocytes of buffalo exhibit relatively higher thermal adaptation than bovine cells.

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.

Immune cell composition of the bronchoalveolar lavage fluid in healthy and respiratory diseased dromedary camels.

BACKGROUND: Respiratory diseases are among the most common and expensive to treat diseases in camels with a great economic impact on camel health, welfare, and production. Bronchoalveolar lavage fluid (BALF) has been proven as a valuable sample for investigating the leukocyte populations in the respiratory tract of several species. In the present study, fluorescent antibody labeling and flow cytometry were used to study the immune cell composition of BALF in dromedary camels. Animals with clinical respiratory diseases (n = seven) were compared with apparently healthy animals (n = 10). In addition, blood leukocytes from the same animals were stained in parallel with the same antibodies and analyzed by flow cytometry. RESULTS: Camel BALF macrophages, granulocytes, monocytes, and lymphocytes were identified based on their forward and side scatter properties. The expression pattern of the cell markers CD172a, CD14, CD163, and MHCII molecules on BALF cells indicates a similar phenotype for camel, bovine, and porcine BALF myeloid cells. The comparison between camels with respiratory disease and healthy camels regarding cellular composition in their BALF revealed a higher total cell count, a higher fraction of granulocytes, and a lower fraction of macrophages in diseased than healthy camels. Within the lymphocyte population, the percentages of helper T cells and B cells were also higher in diseased than healthy camels. The elevated expression of the activation marker CD11a on helper T cells of diseased camels is an indication of the expansion of helper T cells population due to infection and exposure to respiratory pathogens. The higher abundance of MHCII molecules on BALF macrophages from diseased camels indicates a polarization toward an inflammatory macrophage phenotype (M1) in respiratory diseased camels. No significant differences were observed in the systemic leukogram between healthy and diseased animals. CONCLUSIONS: Collectively, the current study represents the first report on flow cytometric analysis of immune cell composition of bronchoalveolar lavage fluid (BALF) in dromedary camels.

Flow Cytometric Analysis of Leukocyte Populations in the Lung Tissue of Dromedary Camels.

Respiratory tract infections are among the most common infections in dromedary camels, with a high impact on animal health, production, and welfare. Tissue-specific distribution of immune cells is one of the important factors that influence the nature and outcome of the immune response to pathogens. Several protocols have recently been described for the flow cytometric analysis of immune cells in the lung tissue of several species. However, no such protocol currently exists for dromedary camels. The aim of the present study was, therefore, to establish a flow cytometric protocol for the identification of immune cell populations in the camel lung tissue and the evaluation of some of their phenotypic and functional properties. Combined staining of camel lung leukocytes with monoclonal antibodies to the pan-leukocyte marker CD45 and the myeloid cell marker CD172a allowed the identification of myeloid cells (CD45+CD172a+) and lymphoid cells (CD45+CD172a-) in the lung of healthy camels. The cell adhesion molecules CD11a and CD18 were found in a higher abundance on myeloid cells compared to lymphoid cells. Based on their differential expression of the LPS receptor CD14, macrophages (CD172a+CD14high cells) were identified as the most abundant immune cell population in the camel lung tissue. In contrast to their dominance in camel peripheral blood, granulocytes (CD172a+CD14low) presented only a minor population in the lung tissue. The higher frequency of γδ T cells in the lung tissue than in peripheral blood suggests a role for these cells in the pulmonary immune system. Flow cytometric analysis of bacterial phagocytosis and ROS production upon bacterial stimulation revealed high antimicrobial activity of camel lung phagocytes, which was comparable with the antimicrobial activity of blood granulocytes. Comparative analysis of immune cell distribution between the cranial and caudal lobes of the camel lung revealed a higher frequency of granulocytes and a lower frequency of macrophages in the cranial compared to the caudal lung lobe. In addition, the higher frequency of cells expressing the M2 macrophage marker CD163 in the caudal lung tissue, with a slightly higher fraction of MHCII-positive cells (M1 phenotype) in the cranial lung tissue, may suggest the distribution of different macrophage subtypes in the different lobes of the camel lung. Such differences between lung lobes could influence the effectiveness of the immune response to infection or vaccination with respiratory pathogens. Collectively, the present study identified some similarities and differences between camels and other farm animals regarding the distribution of the main immune cell populations in their lungs. Further studies are required for comprehensive immunophenotyping of the cellular pulmonary immune system in camels.

Milk Immune Cell Composition in Dromedary Camels With Subclinical Mastitis.

Mastitis represents one of the most important infectious diseases in camels with heavy economic losses due to reduced milk quantity and quality. Balanced immune cell composition and function in the mammary gland are essential for effective immune response to mastitis pathogens. The objective of the present study was to characterize the cellular immune response to subclinical mastitis in the mammary gland of dromedary camels. Therefore, immunostaining and flow cytometry were used to compare the cellular composition, leukocyte phenotype, and cell viability in camel milk from healthy she-camels (n = 8) and she-camels with subclinical mastitis (SCM; n = 6). In addition, the ex vivo phagocytic activity of milk phagocytes was compared between healthy and affected animals. The health status of the mammary gland was evaluated based on the California Mastitis Test (CMT) score. SCM (CMT score of ≥3 in the absence of clinical signs of mastitis) was found in six of the 56 sampled quarters (10.7 %) with only one affected quarter per animal. In comparison to milk from healthy camels, milk from SCM animals showed higher somatic cell count (SCC), higher numbers of CD45+ leukocytes with an expanded fraction of CD172a+ myeloid cells. Within the myeloid cell population, there was an increase in the percentage of granulocytes (CD172a+CD14low) with a decreased percentage of macrophages (CD172a+CD14high) in milk from affected animals compared to healthy animals. The decrease in lymphoid cells in SCM milk was mainly due to the decreased fraction of CD4+ helper T cells. Camel SCM was also associated with a stimulated phenotype, increased cell viability, and enhanced phagocytic activity of the milk phagocytes, macrophages and granulocytes. Collectively, the present study identified significant changes in SCC, leukocyte count, phenotype, viability, and function in association with subclinical mastitis in camels. The results of the present study support a better understanding of host-pathogen interaction mechanisms in the camel mammary gland.

Low levels of soluble DPP4 among Saudis may have constituted a risk factor for MERS endemicity.

Most of the cases of Middle East respiratory syndrome coronavirus (MERS-CoV) were reported in Saudi Arabia. Dipeptidyl peptidase-4 (DPP4) was identified as the receptor for the virus. The level of soluble DPP4 (sDPP4) was found to be reduced in MERS-CoV infected patients while high levels of sDPP4 were suggested to be protective against MERS-CoV in animal models. We investigated whether the Saudi population has lower levels of sDPP4 which makes them more susceptible to MERS-CoV infection and, therefore, could explain the larger number of cases from the country. Blood samples were collected from 219 Saudi blood donors and 200 blood donors from other ethnic groups. The plasma level of sDPP4 was measured by ELISA and the following SNPs in the DPP4 gene; rs35128070, rs1861978, rs79700168, and rs17574, were genotyped by TaqMan SNP genotyping assay. The average level of plasma sDDP4 was significantly lower in Saudis than other Arabs and non-Arabs (P value 0.0003 and 0.012, respectively). The genotypes AG of rs35128070 and GT of rs1861978 were significantly associated with lower sDPP4 among Saudis (P value 0.002 for each). While both genotypes AA and AG of rs79700168 and rs17574 were associated with significantly lower average sDPP4 level in Saudis compared to other ethnic groups (P value 0.031 and 0.032, and 0.027 and 0.014, respectively). Herein, we report that the Saudi population has lower levels of plasma sDPP4 than other ethnic groups, which is associated with genetic variants in the DPP4 gene. This may have contributed to increase the susceptibility of the Saudi population to MERS-CoV infection and could be a factor in the long-lasting persistence of the virus in the country.

The Impact of Anticoagulation Agent on the Composition and Phenotype of Blood Leukocytes in Dromedary Camels.

For the analysis of several cellular biomarkers, blood samples are anticoagulated using different agents with different modes of action. However, for the most commonly used anticoagulants, EDTA and heparin, varying effects on blood components have been reported in different species. As little is known about the impact of anticoagulants on the immunological evaluation of camel leukocytes, the present study analyzed the leukogram, the immunophenotype, and the cell vitality of camel leukocytes separated from blood samples anticoagulated with EDTA or lithium heparin. Using flow cytometry and staining with monoclonal antibodies to several cell surface markers, the composition and immunophenotype of camel leukocytes separated from blood anticoagulated with EDTA or heparin were analyzed. In comparison to EDTA-anticoagulated blood, using lithium heparin as an anticoagulant resulted in reduced numbers of total leukocytes and reduced numbers of neutrophils, which led to a reduced neutrophil to lymphocyte ratio. The analysis of cell necrosis and apoptosis after the staining of leukocytes with the DNA-sensitive dye propidium iodide and the mitochondrial membrane potential probe JC1 revealed a higher fraction of necrotic neutrophils and higher fractions of apoptotic neutrophils and monocytes in heparin blood than in EDTA blood. In addition, monocytes from heparin blood showed higher expression levels of the cell surface markers CD14, CD163, and MHCII when compared to cells from EDTA blood. Similarly, in heparin blood, CD44 and CD172a were expressed higher on neutrophils, while CD11a was expressed higher on lymphocytes in comparison to cells from EDTA blood. The results of the current study indicate the importance of considering the type of anticoagulant when investigating the composition, vitality, and immunophenotype of camel leukocytes.