Toxoplasma gondii and Neospora caninum Antibodies in Dogs and Cats from Egypt and Risk Factor Analysis.

BACKGROUND: Toxoplasma gondii and Neospora caninum are major protozoan parasites of worldwide distribution and significance in veterinary medicine and, for T. gondii, in public health. Cats and dogs, as final hosts for T. gondii and N. caninum, respectively, have a key function in environmental contamination with oocysts and, thus, in parasite transmission. Very little is known about the prevalence of T. gondii infections in dogs and cats in Egypt, and even less about the prevalence of N. caninum in the same hosts. METHODS: In the current study, 223 serum samples of both dogs (n = 172) and cats (n = 51) were investigated for specific antibodies to T. gondii and N. caninum using commercially available ELISAs. A risk factor analysis was conducted to identify factors associated with seropositivity. RESULTS & DISCUSSION: Exposure to T. gondii was reported in 23.3% of the dogs and in 9.8% of the cats, respectively. In addition, N. caninum-specific antibodies were recorded in 5.8% of dogs and in 3.4% of cats. A mixed infection was found in two dogs (1.2%) and in one cat (2%). Antibodies to T. gondii in dogs were significantly more frequent in dogs aged 3 years or more and in male German Shepherds. As this breed is often used as watchdogs and was the most sampled breed in Alexandria governorate, the purpose "watchdog" (compared to "stray" or "companion"), the male sex, and the governorate "Alexandria" also had a significantly higher seroprevalence for T. gondii. No factors associated with antibodies to N. caninum could be identified in dogs, and no significant factors were determined in cats for either T. gondii or N. caninum infection. Our study substantially adds to the knowledge of T. gondii infection in dogs and cats and presents data on N. caninum infection in cats for the first and in dogs in Egypt for the second time.

Co-circulation of GI.1 and GI.2 genotypes of rabbit hemorrhagic disease virus in Egypt.

Recently, Egypt has experienced an increased incidence of rabbit hemorrhagic disease virus (RHDV) infection even among vaccinated rabbits. The present study estimates the emergence of RHDV in vaccinated (n = 10) and unvaccinated (n = 8) domestic rabbitries in Beheira and Kafr El-Sheikh provinces, Egypt, during the period 2018-2020. A total of 8 out of 18 (44.4%) liver extracts were able to agglutinate human type O RBCs with HA titers ranged from 8 to 12 log2, and then subsequently confirmed for the presence of RHDV RNA using a reverse transcriptase-polymerase chain reaction (RT-PCR). The VP60 gene sequences of three selected isolates, designated Beh-1, Beh-9 and kaf-14, were submitted to the GenBank database and the accession numbers MZ782083 to MZ782085 were assigned, respectively. Phylogenetic analysis revealed that the Kaf-14 isolate was placed into the GI.1 genotype, while the Beh-1 and Beh-9 isolates were grouped into the GI.2 genotype. Overall, the three isolates shared 78.6-98.7%.nucleotide identity with previously published Egyptian sequences. In comparison with the GI.1a Giza2006 vaccine strain, the three isolates exhibited divergence ranging from 4.5 to 17.4% at the amino acid level. Approximately 55.5-87.5% of the amino acid substitutions were located in the P2 subdomain of the VP60 capsid protein which contains the main determinants of antigenicity and cellular recognition. In conclusion, our results provide crucial evidence for the co-circulation of RHDV GI.1 and GI.2 genotypes in Egypt and highlight the antigenic diversity among vaccine and field strains. Therefore, new effective vaccines are urgently required to counter the spread of GI.1 and GI.2 genotypes in Egypt.

Cellular infiltration, cytokines, and histopathology of skin lesions associated with different clinical forms and stages of naturally occurring lumpy skin disease in cattle.

Lumpy skin disease (LSD) caused by the Capripoxvirus LSD virus which infects cattle, leading to a serious disease characterized by fever and the eruption of skin nodules all over the surface of the body. Our understanding of the pathogenesis of this disease is still incomplete, particularly the immunopathological alterations occurring in the skin nodules of infected animals. Therefore, we collected skin nodules from naturally infected cattle with different forms of the disease, both in the early stage of clinical infection and after disease progression. The skin samples were examined both histopathologically and immunohistochemically using a variety of antibodies targeting immune cellular markers and cytokines. As a result, the dermatohistopathology revealed orthokeratotic hyperkeratosis, vasculitis, epidermal microvesicles, and cellules claveleuses of Borrel in the early stage of infection, with the severity of the lesions correlating with the severity of the clinical disease. Meanwhile, late-stage samples had epidermal hyperkeratosis as well as dermal lymphocytic and histiocytic infiltrations. The predominant cellular infiltrates in the cutaneous lesions of early-stage LSD samples were interferon (IFN)-γ+ cells and CD4+ T lymphocytes with few macrophage lineage cells. However, in the late-stage samples, numerous Iba-1+ macrophages, with few IFN-γ+ cells and CD4+ T lymphocytes, were detected. Our findings indicate that IFN-γ+ cells, CD4+ T lymphocytes, and macrophages play a key role in the immunity against natural LSD virus infection and imply that cutaneous vasculopathy associated with LSD virus infection is an immune-mediated lesion. The current study contributes to our understanding of the pathogenesis of LSD.

CXCR6+CD4+ T cells promote mortality during Trypanosoma brucei infection.

Liver macrophages internalize circulating bloodborne parasites. It remains poorly understood how this process affects the fate of the macrophages and T cell responses in the liver. Here, we report that infection by Trypanosoma brucei induced depletion of macrophages in the liver, leading to the repopulation of CXCL16-secreting intrahepatic macrophages, associated with substantial accumulation of CXCR6+CD4+ T cells in the liver. Interestingly, disruption of CXCR6 signaling did not affect control of the parasitemia, but significantly enhanced the survival of infected mice, associated with reduced inflammation and liver injury. Infected CXCR6 deficient mice displayed a reduced accumulation of CD4+ T cells in the liver; adoptive transfer experiments suggested that the reduction of CD4+ T cells in the liver was attributed to a cell intrinsic property of CXCR6 deficient CD4+ T cells. Importantly, infected CXCR6 deficient mice receiving wild-type CD4+ T cells survived significantly shorter than those receiving CXCR6 deficient CD4+ T cells, demonstrating that CXCR6+CD4+ T cells promote the mortality. We conclude that infection of T. brucei leads to depletion and repopulation of liver macrophages, associated with a substantial influx of CXCR6+CD4+ T cells that mediates mortality.

Prevalence of tick-borne haemoparasites and their perceived co-occurrences with viral outbreaks of FMD and LSD and their associated factors.

Species of Theileria, Babesia, and Anaplasma are Tick-borne pathogens (TBPs) that are prevalent throughout the world, particularly in the tropical and subtropical regions. Associated diseases of Theileriosis, Babesiosis, and Anaplasmosis, respectively, represents a major threat to livestock production in many countries. TBPs have a high prevalence in different geographical locations in Egypt. Foot and mouth disease (FMD) and Lumpy skin disease (LSD) are considered endemic bovine viral diseases in Egypt. Our clinical observations during the epidemics of LSD and FMD viruses showed higher prevalence rates for the TBPs. To investigate this correlation, a total of 670 samples from cattle and buffalo were collected during the summers of 2017 and 2018 distributed throughout ranches and smallholders in two geographical locations in Egypt. Two farms with a recent clinical outbreak of LSD with a total of 270 animals, while the other location included three farms with a recent FMD outbreak with a combined 400 cattle. Examined animals were classified mainly according to age, gender, species, breed (native versus crossbred), and the presence of ticks. Whole blood samples were collected for TBPs and viral (LSD and FMD) examinations, while tissue specimens were collected for detection of FMD and LSD viruses by real-time PCR. Our results confirmed significantly higher prevalence rates for the TBPs in LSD-positive than LSD-negative animals, while no significant difference could be detected for the prevalence rate of the TBPs in the FMD positive and negative groups. The prevalence of Babesia and Theileria was significantly (P < 0.05) higher in cross-breeds than native cattle. Infections with Anaplasma and co-infections with Babesia-Anaplasma and Theileria-Anaplasma were significantly higher in native than cross-breeds cattle. The intensity of parasitic infection (parasitemia) has a significant difference in the positive groups for the two viruses compared to the negative groups. These results collectively confirming the enhancing role of LSD on the prevalence rate of the haemoprotozoal infections leading to more serious outcomes to the livestock infections, and therefore the control of haemoprotozoal infections should be implemented as a part of viral epidemics control.

IL-27 Negatively Regulates Tip-DC Development during Infection.

Tumor necrosis factor (TNF)/inducible nitric oxide synthase (iNOS)-producing dendritic cells (Tip-DCs) have profound impacts on host immune responses during infections. The mechanisms regulating Tip-DC development remain largely unknown. Here, using a mouse model of infection with African trypanosomes, we show that a deficiency in interleukin-27 receptor (IL-27R) signaling results in escalated intrahepatic accumulation of Ly6C-positive (Ly6C+) monocytes and their differentiation into Tip-DCs. Blocking Tip-DC development significantly ameliorates liver injury and increases the survival of infected IL-27R-/- mice. Mechanistically, Ly6C+ monocyte differentiation into pathogenic Tip-DCs in infected IL-27R-/- mice is driven by a CD4+ T cell-interferon gamma (IFN-γ) axis via cell-intrinsic IFN-γ signaling. In parallel, hyperactive IFN-γ signaling induces cell death of Ly6C-negative (Ly6C-) monocytes in a cell-intrinsic manner, which in turn aggravates the development of pathogenic Tip-DCs due to the loss of the negative regulation of Ly6C- monocytes on Ly6C+ monocyte differentiation into Tip-DCs. Thus, IL-27 inhibits the dual-track exacerbation of Tip-DC development induced by a CD4+ T cell-IFN-γ axis. We conclude that IL-27 negatively regulates Tip-DC development by preventing the cell-intrinsic effects of IFN-γ and that the regulation involves CD4+ T cells and Ly6C- monocytes. Targeting IL-27 signaling may manipulate Tip-DC development for therapeutic intervention.IMPORTANCE TNF/iNOS-producing dendritic cells (Tip-DCs) are at the front line as immune effector cells to fight off a broad range of invading microbes. Excessive development of Tip-DCs contributes to tissue destruction. Thus, identifying master regulators of Tip-DC development is fundamental for developing new therapeutic strategies. Here, we identify Tip-DCs as a terminal target of IL-27, which prevents Tip-DC-mediated early mortality during parasitic infections. We demonstrate that IL-27 inhibits Tip-DC development via a dual-track mechanism involving the complex interactions of effector CD4+ T cells, Ly6C- monocytes, and Ly6C+ monocytes. These findings delineate an in-depth view of mechanisms of Tip-DC differentiation that may have significant implications for the ongoing development of IL-27-based immunotherapy.

Comparative Study of the Pathogenesis of Rhinopneumonitis Induced by Intranasal Inoculation of Hamsters with Equine Herpesvirus-9, Equine Herpesvirus-1 strain Ab4p and Zebra-borne Equine Herpesvirus-1.

Equine herpesvirus-9 (EHV-9), equine herpesvirus-1 (EHV-1) and zebra-borne EHV-1 are members of the family Herpesviridae and cause encephalitis and rhinopneumonitis in a range of animal species. The aim of this study was to characterize and compare the rhinopneumonitis induced by experimental intranasal inoculation of groups of hamsters with EHV-9, EHV-1 strain Ab4p or zebra-borne EHV-1 viruses. Animals inoculated with EHV-9 had earlier and more severe neurological and respiratory signs than those inoculated with EHV-1 strain Ab4p or zebra-borne EHV-1. At 4-5 days post inoculation (dpi), hamsters inoculated with EHV-9 had significantly increased expression of open reading fame (ORF) 30, the viral gene encoding the DNA polymerase, in lung tissue. ORF 30 expression at these time points was higher in the hamsters infected with EHV-9 than in those inoculated with the other two viruses. Severe, mild or very mild rhinitis was seen in animals inoculated with EHV-1 strain Ab4p, EHV-9 and zebra-borne EHV-1, respectively. Viral antigen was detected in olfactory receptor neurons, inflammatory cells and desquamated epithelial cells in animals in all groups until 5 dpi. Tracheitis was also seen in all three virus-infected groups with viral antigen detected in tracheal epithelium. Inoculated hamsters developed interstitial pneumonia of increasing severity over the course of the experiment. Bronchopneumonia and vasculitis were also seen in all three infected groups. These results confirm that, in addition to their neurotropism, EHV-9 and zebra-borne EHV-1 are pneumotropic viruses. EHV-1 strain Ab4p caused more severe upper respiratory tract disease, but no significant differences were detected in the severity of pneumonia induced by each virus.

Correlation of Immunomodulatory Cytokine Expression with Histopathological Changes and Viral Antigen in a Hamster Model of Equine Herpesvirus-9 Encephalitis.

A group of hamsters (n = 25) was intranasally infected with equine herpesvirus-9 (EHV-9) and mRNA transcription levels of several proinflammatory (IFN-γ, TNF-α and IL-6) and anti-inflammatory (IL-4, IL-10 and TGF-ß) cytokines were investigated in brain tissue using RT-qPCR. These levels were correlated with the severity of sequential histopathological changes and intensity of immunohistochemical labelling of virus antigen in brain. Early and progressive upregulation of all the proinflammatory and anti-inflammatory cytokines investigated (P < 0.05) was correlated with increasing severity of encephalitis and viral antigen expression from 2 days post infection (dpi) with a peak at 4-5 dpi (P <0.05).

Time Course-Dependent Study on Equine Herpes Virus 9-Induced Abortion in Syrian Hamsters.

This study aimed to follow the time-course pathogenesis of EHV-9 abortion in early and late trimesters. Twenty-seven pregnant hamster dams were divided into three groups: (G1) control, (G2) EHV-9-inoculated on the 5th day (early trimester), and (G3) EHV-9-inoculated on the 10th day of gestation (late trimester). Dams were sacrificed at different time points during gestation and examined for viremia and viral DNA in different fetal and maternal tissues and pathological changes in fetal tissue, placenta, and cytokines. Animals in G3 showed a marked increase in the number of dead fetuses than those in G2. Histopathological findings of G2 showed early band coagulative necrosis of maternal spaces and stromal decidual cells. Necrotic changes were observed within the decidua basalis, spongiotrophoblast layer, and labyrinth. First, the virus was localized within mononuclear leukocytes in the decidua capsularis and basalis, and within the necrotic chorionic villi and cervical epithelium. G3 demonstrated degenerative changes within the chorionic villi and trophospongium. The virus antigen was observed within the chorionic villi, trophoblasts, mononuclear cells, and fetal tissues. In conclusion, EHV-9 induced abortion mostly occurs through necrosis of the chorionic villi and cannot cross through the capsular placenta in the early trimester but can through the developed decidual placentation.

First report of seroprevalence and genetic characterization of avian orthoreovirus in Egypt.

Recently, the Egyptian broiler industry has experienced an increased incidence of avian reovirus (ARV) infections. However, to date, no studies have been carried out to investigate the epidemiologic status of ARV infections as well as the genetic characteristics of the currently circulating ARV strains. The present study estimates the seroprevalence of ARV infections in Alexandria, El-Behera, Giza, Kafr El-Sheikh, and Gharbia governorates, Egypt, during the period 2017-2018. A total of 150 serum samples from 15 unvaccinated broiler flocks with suspicious ARV infection were screened using a commercial enzyme-linked immunosorbent assay kit. All the tested flocks were found to be positive for ARV-specific antibodies, and the overall seropositivity rate was 80.6%. Meanwhile, 5 (33.3%) flocks were confirmed for the presence of ARV through a reverse transcription-polymerase chain reaction (RT-PCR) assay based on the σA-encoding gene. Phylogenetic analysis based on the nucleotide sequences of the σA-encoding gene revealed that the obtained ARV isolate, designated EGY1, was grouped in the S1113-like cluster of ARV and displayed 100% and 98.7% nucleotide identity with the Chinese MSO1 isolate and the S1133 vaccine strain, respectively. In addition, amino acid alignments with the S1133 vaccine strain revealed that the σA protein of the EGY1 isolate carried the substitutions G81S and A118V. In conclusion, the present study provides the evidence for a ubiquitous distribution of ARV infection in Egypt as well as represents a starting point for genetic characterization of the currently circulating ARV strains.

The first identification of contagious caprine pleuropneumonia (CCPP) in sheep and goats in Egypt: molecular and pathological characterization.

Contagious caprine pleuropneumonia (CCPP) is one of the most fatal and contagious diseases of goats. To date, the occurrence of CCPP in Egypt has not been reported. During the period from 2017 to 2018, 200 goats and 400 sheep from Matrouh Governorate (Al Alamein and El Hammam cities) were suspected to have CCPP; animals were examined to confirm the presence of CCPP infection as well as the epidemiological status, clinical features, and molecular and histopathologic characteristics of lung tissues. Additionally, a treatment trial was performed to assess the efficacy of anti-mycoplasma therapy in the treatment of clinical cases of this disease. The occurrence of CCPP was 32.5% and 5% in goats and sheep, respectively, while case fatality was 30% and 8% in goats and sheep, respectively. The clinical forms of CCPP in both sheep and goats varied from per-acute to acute or chronic cases. Histopathological analysis of lung tissues from dead cases (either sheep or goats) revealed different stages of broncho- and pleuropneumonia ranging from per-acute to acute or chronic stages. Lung tissues showed severe congestion of interalveolar capillaries, flooding of alveoli and bronchi with a fibrinous exudate, a high degree of pleural thickening, and multifocal areas of necrosis that were sometimes sequestered in the fibrous capsule. Isolation of Mycoplasma capricolum subspecies capripneumoniae (Mccp) was confirmed in all dead cases by agar and broth culture methods and polymerase chain reaction. The treatment trial revealed that the marbofloxacin and spiramycin groups had a higher cure rate (70%) than the oxytetracycline group (40%) and a lower fatality rate (30%) than the oxytetracycline group (60%). Conclusively, infection with CCPP in goats and sheep is considered to be novel for Mccp in Egypt, where this species is considered to be the main pathogen in goats, not in sheep. Additionally, it could be concluded that treatment may be effective only if given early. Further comprehensive surveys are required to investigate the risk of CCPP in goats and sheep in all Egyptian governorates.

Protection conferred by a vaccine derived from an inactivated Egyptian variant of infectious bronchitis virus: a challenge experiment.

The current study investigated the protective efficacy of a formalin-inactivated infectious bronchitis virus (IBV) vaccine derived from the field strain KP729422, which exhibits low S1 spike protein sequence homology (77.1-79.8%) with the currently used vaccine strains in Egypt. Two-week-old, specific-pathogen-free chickens were subcutaneously inoculated with a single dose of the vaccine containing 106.7 50% embryo infective dose (EID50) of the inactivated virus. At 6 weeks of age, the chickens were challenged with 104 EID50 of the same virus strain via the oculonasal route. In comparison with the unvaccinated challenged group, the vaccinated chickens had significantly higher IBV-neutralizing antibody titers and exhibited efficient protection against challenge on the basis of tracheal ciliary activity. However, the challenge virus was recovered from the kidneys and tracheas of these chickens at rates of 40% and 60%, respectively. These findings suggest that a single application of the vaccine may provide sufficient clinical and respiratory protection, but may not ensure complete protection against infection by the challenge virus.

Lesions and Distribution of Viral Antigen in the Brain of Hamsters Infected With Equine Herpesvirus (EHV)-9, EHV-1 Strain Ab4p, and Zebra-Borne EHV-1.

Encephalitis in hamsters, which was induced by equine herpesvirus (EHV)-9, EHV-1 strain Ab4p, and zebra-borne EHV-1, was investigated and compared to assess viral kinetics and identify the progression and severity of neuropathological findings. Hamsters were inoculated with EHV-9, EHV-1 strain Ab4p, and zebra-borne EHV-1 via the nasal route and euthanized at 24, 48, 72, 96, 120, 144, and 168 hours postinoculation (HPI). The inoculated hamsters had mild to severe neurological signs at 60 to 72, 96, and 120 HPI, and the mortality rate was 75%, 0%, and 0% for animals inoculated with EHV-9, EHV-1 strain Ab4p, and zebra-borne EHV-1 viruses, respectively. Inoculated hamsters had varying degrees of rhinitis and lymphoplasmacytic meningoencephalitis, as well as differences in the severity and distribution of cerebral lesions. Furthermore, the cellular distribution of viral antigen depended on the inoculated virus. Neuronal necrosis was widely detected in animals inoculated with EHV-9, while marked perivascular cuffs of infiltrating inflammatory cells and gliosis were detected in animals inoculated with EHV-1 strain Ab4p and zebra-borne EHV-1. In the present study, 3 viruses belonging to the herpesvirus family induced encephalitis after initial propagation in the nasal cavity. These viruses might travel to the brain via the olfactory pathway and/or trigeminal nerve, showing different distributions and severities of neuropathological changes.

Effect of a single point mutation on equine herpes virus 9 (EHV-9) neuropathogenicity after intranasal inoculation in a hamster model.

This study aimed to investigate the neuropathogenesis of equine herpes virus 9 (EHV-9) by studying the effects of a single point mutation introduced in two different EHV-9 genes. The two EHV-9 mutants, 14R and 19R, were generated carrying a point mutation in two separate EHV-9 genes. These mutants, along with the wild-type EHV-9, were used to infect a hamster model. The EHV-9- and 19R-infected groups showed earlier and more severe clinical signs of infection than the 14R-infected group. The white blood cells (WBCs) count was significantly increased in both EHV-9- and 19R-infected groups compared to the 14R-infected group at the 4th day post infection (DPI). Viremia was also detected earlier in both EHV-9- and 19R-infected groups than 14R-infected group. There were differences in the anterograde transmission pattern of both EHV-9 and 19R compared to 14R inside the brain. Serum TNF-α, IL-6 and IFN-γ levels were significantly increased in both EHV-9- and 19R-infected groups compared to the 14R-infected group. Histopathological and immunohistochemical analyses revealed that the mean group scores for the entire brain were significantly higher in both EHV-9- and 19R- infected groups than 14R-infected group. Collectively, these results confirm that the gene product of Open Reading Frame 19 (ORF19) plays an important role in EHV-9 neuropathogenicity and that the mutation in ORF19 is responsible for the attenuation of EHV-9.

Clinical Spectrum of Cerebral Palsy and Associated Disability in South Egypt: A Local Survey Study.

BACKGROUND: Cerebral palsy is the most common cause of motor disability in children with a prevalence of 2-10/1,000 live births in the developing areas. AIM: The epidemiology, clinical picture, and associated comorbidities in CP have been extensively studied in high-resource countries, but in low-resource areas, including Africa, those studies are still lacking. METHODS: Cerebral palsy cases were prospectively recruited from every physiotherapy centre in Bani-Mazar city, Egypt, in a cross-sectional study from May 2015 to November 2015. RESULTS: Two hundred cases were enrolled with a prevalence of 1 per 1000 live births. Within the study population, 72.5% were the spastic type, 16% were dyskinetic, 7% were ataxic, and 4.5% were hypotonic. The most common comorbidities were cognitive impairment and epilepsy affecting 77% and 38%, respectively. CONCLUSION: Cerebral palsy in developing countries has a higher prevalence and different clinical profile regarding severity and associated disability. The perinatal and high-quality neonatal care together with physical therapy and rehabilitation programs is still lacking in developing countries.