Days Post-infection Research Articles

Effectiveness of Povidone-Iodine 1% Eye Drop on Streptococcus pneumoniae and Escherichia coli Induced-Keratitis in Mice

Background: Bacterial keratitis is an ophthalmic infection that may result in irreversible corneal damage. This study aimed to examine the effectiveness of povidone-iodine eye drop 1% in eye infection caused by inoculation of Streptococcus pneumoniae and Escherichia coli of mice. Materials and Methods: In this study, 49 adult male CBA/J mice were used that divided into seven equal groups. The corneas of all mice were scratched and infected with a clinical strain of either S. pneumoniae or E. coli topically, except control group. Subgroups received chloramphenicol 0.5% eye drop twice daily in case of S. pneumoniae infection or ciprofloxacin 0.3% eye drop every 4 hours following E. coli infection from or povidone-iodine 1% eye drop in both groups, from post infection (PI) day 3 to7. Slit lamp examinations (SLE) of the corneas and eyes were performed every day to examine detectable or intense corneal opacity and erosion. Results: In all infected mice, SLE scores were significantly higher than the control group on PI day 3. Scores increased steadily by time in all infected groups without treatment, reached to maximal value on PI day 7. In infected groups, treatment with either povidone-iodine 1% or chloramphenicol 0.5% or ciprofloxacin 0.3% on day 3, significantly decreased the SLE scores on PI day 7. Conclusion: Povidone-Iodine 1% was effective to decrease S. pneumoniae and E. coli induced-keratitis symptoms in mice. Treatment with povidone-iodine 1% was observed time-dependently and was comparable to common eye drop antibiotics. [GMJ.2019;8:e1161]

Effectiveness of Povidone-Iodine 1% Eye Drop on Streptococcus pneumoniae and Escherichia coli Induced-Keratitis in Mice.

Background: Bacterial keratitis is an ophthalmic infection that may result in irreversible corneal damage. This study aimed to examine the effectiveness of povidone-iodine eye drop 1% in eye infection caused by inoculation of Streptococcus pneumoniae and Escherichia coli of mice. Materials and Methods: In this study, 49 adult male CBA/J mice were used that divided into seven equal groups. The corneas of all mice were scratched and infected with a clinical strain of either S. pneumoniae or E. coli topically, except control group. Subgroups received chloramphenicol 0.5% eye drop twice daily in case of S. pneumoniae infection or ciprofloxacin 0.3% eye drop every 4 hours following E. coli infection from or povidone-iodine 1% eye drop in both groups, from post infection (PI) day 3 to7. Slit lamp examinations (SLE) of the corneas and eyes were performed every day to examine detectable or intense corneal opacity and erosion.Results: In all infected mice, SLE scores were significantly higher than the control group on PI day 3. Scores increased steadily by time in all infected groups without treatment, reached to maximal value on PI day 7. In infected groups, treatment with either povidone-iodine 1% or chloramphenicol 0.5% or ciprofloxacin 0.3% on day 3, significantly decreased the SLE scores on PI day 7. Conclusion: Povidone-Iodine 1% was effective to decrease S. pneumoniae and E. coli induced-keratitis symptoms in mice. Treatment with povidone-iodine 1% was observed time-dependently and was comparable to common eye drop antibiotics.

Increased Innate Lymphoid Cell 3 and IL-17 Production in Mouse Lamina Propria Stimulated with Giardia lamblia.

Innate lymphoid cells (ILCs) are key players during an immune response at the mucosal surfaces, such as lung, skin, and gastrointestinal tract. Giardia lamblia is an extracellular protozoan pathogen that inhabits the human small intestine. In this study, ILCs prepared from the lamina propria of mouse small intestine were incubated with G. lamblia trophozoites. Transcriptional changes in G. lamblia-exposed ILCs resulted in identification of activation of several immune pathways. Secretion of interleukin (IL)-17A, IL-17F, IL-1β, and interferon-γ was increased, whereas levels of IL-13, IL-5, and IL-22, was maintained or reduced upon exposure to G. lamblia. Goup 3 ILC (ILC3) was found to be dominant amongst the ILCs, and increased significantly upon co-cultivation with G. lamblia trophozoites. Oral inoculation of G. lamblia trophozoites into mice resulted in their presence in the small intestine, of which, the highest number of parasites was detected at the 5 days-post infection. Increased ILC3 was observed amongst the ILC population at the 5 days-post infection. These findings indicate that ILC3 from the lamina propria secretes IL-17 in response to G. lamblia, leading to the intestinal pathology observed in giardiasis.

Comparative study on hemato-biochemical alterations and selected acute phase protein response in native sheep experimentally infected with bluetongue virus serotypes 10 and 24

Bluetongue (BT) is an infectious, non-contagious, insect-transmitted viral disease of sheep, other domestic, and wild ruminants caused by bluetongue virus (BTV), a prototype species of the genus Orbivirus of the family Reoviridae. The study was designed to determine the hematological and biochemical alterations and acute phase protein response in BTV sero-negative sheep experimentally infected with bluetongue virus serotype-10 and 24. The BTV-infected group comprising of 18 animals, 6 each inoculated with 6 ml of clarified virus containing 1 × 106/ml TCID50 of BTV-10 or 24 by intradermal route, 6 animals inoculated with equal volumes of BTV-10 and 24 and 4 animals with 6 ml of mock infected culture fluid served as uninfected control group. The blood and serum samples were analyzed at 0, 1, 3, 7, 11, and 16 days post-infection (DPI). Hematological findings in this study showed a significant decrease in the total number of white blood cells in BTV-10 infected group, a significant increase in circulating neutrophils decrease in lymphocytes in all the infected groups, in PCV in BTV-24 and co-infected groups, and RBC count in only BTV-24-infected group at 11 DPI. The results of serum biochemical analyses of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) exhibited increased levels in BTV-24 and co-infected animals. A significant rise in the serum level of creatine kinase (CK) was observed from 3 DPI in the infected groups. Serum levels of total proteins decreased significantly at 11 DPI in BTV-24 infected animals. Significant increase in CRP and fibrinogen levels was observed at 7 and 11 DPI in the infected groups. The significantly decreased levels of TLC, hemoglobin, PCV, CK, and acute phase proteins such as fibrinogen and CRP indicated the inflammatory changes associated with the virus replication and the consequent vascular damage and inflammatory changes in the tissues. Further, the variations in the hemato-biochemical and acute phase response observed between the two serotypes BTV-10 and 24 probes to the difference in virulence and pathogenesis of the serotypes in infected sheep.

Virulence evaluation of classical swine fever virus subgenotype 2.1 and 2.2 isolates circulating in China

Classical swine fever (CSF) remains an important pig disease in China, where it usually presents with mild or atypical clinical manifestations, with large scale outbreaks rarely seen. This has led to speculation about the possible circulation of viral strains of low virulence. To investigate this possibility, five field isolates within the predominant genotype 2 (2.1b, 2.1c, 2.1 h and 2.2) were evaluated and compared by experimental infection of naturally farrowed but colostrum-deprived piglets. All infected piglets displayed clinical signs, including persistent high fever, depression, anorexia, dyspnea, conjunctivitis, constipation, and hesitant gait. Typical pathological lesions, including pulmonary edema, hemorrhagic or cellulosic exudation, and swelling and hemorrhage of lymph nodes, were observed. Viremia and Erns protein expression in the blood of all infected animals were detectable from 3 to 5 days post infection (DPI), their presence correlating with the onset of fever, clinical signs and leukopenia. E2 antibody did not develop in any of the field CSFV-infected piglets during the disease course, while Erns antibody was detectable in 4–56% of infected animals at various time points. Mortalities ranged from 20 to 80% within 21 DPI, progressing to 100% by 43 DPI. Based on clinical scores and fatalities within 21 DPI, 2 of the 5 field isolates were classified as of moderate virulence and 3 of high virulence; i.e., no field isolates of low virulence were identified. The study has provided data supporting the use of these isolates as challenge viruses to evaluate the efficacy of current CSF vaccines.

Cryptosporidium proventriculi sp. n. (Apicomplexa: Cryptosporidiidae) in Psittaciformes birds

Cryptosporidiosis is a common parasitic infection in birds that is caused by more than 25 Cryptosporidium species and genotypes. Many of the genotypes that cause avian cryptosporidiosis are poorly characterized. The genetic and biological characteristics of avian genotype III are described here and these data support the establishment of a new species, Cryptosporidium proventriculi. Faecal samples from the orders Passeriformes and Psittaciformes were screened for the presence of Cryptosporidium by microscopy and sequencing, and infections were detected in 10 of 98 Passeriformes and in 27 of 402 Psittaciformes. Cryptosporidium baileyi was detected in both orders. Cryptosporidium galli and avian genotype I were found in Passeriformes, and C. avium and C. proventriculi were found in Psittaciformes. Cryptosporidium proventriculi was infectious for cockatiels under experimental conditions, with a prepatent period of six days post-infection (DPI), but not for budgerigars, chickens or SCID mice. Experimentally infected cockatiels shed oocysts more than 30 DPI, with an infection intensity ranging from 4,000 to 60,000 oocysts per gram (OPG). Naturally infected cockatiels shed oocysts with an infection intensity ranging from 2,000 to 30,000 OPG. Cryptosporidium proventriculi infects the proventriculus and ventriculus, and oocysts measure 7.4 × 5.8 μm. None of the birds infected C. proventriculi developed clinical signs.

Th2-related cytokines are associated with Fasciola gigantica infection and evasion in the natural host, swamp buffalo

The infection of ruminants by Fasciola spp. always induces a non-protective Th2-type immune response. However, little is known about changes in the local and systemic immune environment during F. gigantica migration in buffalo. In this study, native swamp buffaloes were each infected with 500 viable F. gigantica metacercariae. Mesenteric lymph node (MLN), hepatic lymph node (HLN), spleen, and serum samples were collected from control and infected buffaloes at 3, 10, 28, 42, 70, and 98 days post-infection (DPI). The mRNA expression levels of the Th1- and Th2-related cytokines IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, IFN-γ, TNF-α, and CD4 were measured during different infection stages in the MLNs, spleens, and HLNs using quantitative real-time PCR (qRT-PCR). Levels of the specific anti-ESP isotype antibodies IgG, IgG1, and IgG2 were used to reflect changes in humoral immunity. The results of this study indicated that swamp buffaloes were susceptible to F. gigantica infection, and that susceptibility to this infection was closely related to the cytokine environment associated with the Th2-type immune response. The MLNs showed a mixed Th1- and Th2-type immune response during the acute infection stages, after which the production of these cytokines returned to normal. Cytokine expression in the HLNs also expressed a mixed Th1- and Th2-type immune response during the early infection stages. When the infection became chronic, the typical Th2 immune response was induced in the HLNs. At the acute infection stages, the spleen exhibited a Th2 immune response. Nevertheless, cytokines associated with the Th1 and Th2 immune responses were upregulated at 98 DPI. In addition, the total IgG and IgG1 of the parasite-specific antibodies increased. This suggested that the Th2-related cytokines and IgG1 induced by F. gigantica infection might mediate successful F. gigantica infection in the natural host, swamp buffalo.

Biological characterization and pathogenicity of a newly isolated Chinese highly virulent genotype GIIa porcine epidemic diarrhea virus strain.

Since 2010, continual outbreaks of highly virulent variants of porcine epidemic diarrhea virus (PEDV) belonging to genotype GII have led to serious economic losses for the Chinese swine industry. To better understand the biological characteristics and pathogenicity of the current prevalent Chinese PEDV field strains, in this study, a highly virulent Chinese genotype GIIa PEDV strain, CH/HBXT/2018, was isolated and serially propagated using Vero cells. Sequencing and phylogenetic analysis showed that strain CH/HBXT/2018 contained novel insertion and deletion mutations in the S gene region relative to the classical strain and belonged to the genotype GIIa, similar to other recently isolated PEDV strains from China and the United States. Pig infection studies indicated that the CH/HBXT/2018 strain was highly virulent in suckling piglets, and the median pig diarrhea dose (PDD50) was 8.63 log10PDD50/3 mL at 7 days postinfection (DPI). The results of the present study are important for future PEDV challenge studies and the development of new PEDV vaccines based on prevalent field strains for the prevention and control of PED in China.

Death Mechanisms of Pulmonary Alveolocytes in Mice Infected with Influenza Viruses A/H1N1/California/04/2009 and A/H5N1/Goose/Krasnoozerskoye/627/05.

In CBA mice infected with influenza viruses A/H1N1/California/04/2009 and A/H5N1/Goose/Krasnoozerskoye/627/05 in a dose of 10 MLD50, the mechanisms of death of pulmonary alveolocytes over 10 postinfection days were studied by light microscopy, immunohistochemistry, and morphometry. In mice infected with A/H1N1, alveolocytes died predominantly via necrosis, while apoptosis mostly employed the mitochondrial pathway. In mice infected with A/H5N1, apoptosis was the dominant mechanism of alveolocyte death proceeded via membrane receptor signaling followed by switching to FAS-mediated pathway via activation of FADD, the apoptotic signal transduction protein.

Generation of microalga Chlamydomonas reinhardtii expressing shrimp antiviral dsRNA without supplementation of antibiotics

RNA interference (RNAi) is an effective way of combating shrimp viruses by using sequence-specific double-stranded (dsRNA) designed to knock down key viral genes. The aim of this study was to use microalgae expressing antiviral dsRNA as a sustainable feed supplement for shrimp offering viral protection. In this proof of concept, we engineered the chloroplast genome of the green microalga Chlamydomonas reinhardtii for the expression of a dsRNA cassette targeting a shrimp yellow head viral gene. We used a previously described chloroplast transformation approach that allows for the generation of stable, marker-free C. reinhardtii transformants without the supplementation of antibiotics. The generated dsRNA-expressing microalgal strain was then used in a shrimp feeding trial to evaluate the efficiency of the algal RNAi-based vaccine against the virus. Shrimps treated with dsRNA-expressed algal cells prior to YHV infection had 50% survival at 8 day-post infection (dpi), whereas 84.1% mortality was observed in control groups exposed to the YHV virus. RT-PCR using viral specific primers revealed a lower infection rate in dsRNA-expressing algae treated shrimp (55.6 ± 11.1%) compared to control groups (88.9 ± 11.1% and 100.0 ± 0.0%, respectively). Our results are promising for using microalgae as a novel, sustainable alternative as a nutritious, anti-viral protective feedstock in shrimp aquaculture.

Pathogenicity of porcine sapelovirus infection in mice

Porcine Sapelovirus (PSV) is a RNA virus belonging to a new genus Sapelovirus of family Picornaviridae. PSV has been isolated from India in 2016. In the present study, mice experiment was conducted to detect the ability of PSV to infect mice and its ability to induce pathological lesions. The intestinal and extra intestinal spread of the PSV virus in three week-old Swiss albino mice inoculated with PSV virus quantified by probe based real time PCR are described. Herein, three groups were made with 10 mice per group (both sex). The first group was infected through oral route (8×106, TCID50, 240 μl/mice) while the second through intra-peritoneal route (8×106, TCID50, 240 μl/mice) and the third group was inoculated with PBS of neutral pH orally and intra-peritoneal route. Seven mice (each from oral and intraperitoneal route and three from control group were sacrificed at 5th, 7th, 9th, 12th, 15th, 17th, 21st day post infection (DPI). Indian strain was able to replicate in mice organs up to 15 DPI in oral route and 9 DPI in intraperitoneal route. By real-time reverse transcription (RT) PCR, PSV was detected in most of the organs but with highest viral load in the small intestine and large intestine than extra-intestinal organs in the orally infected mice. In addition, this Indian strain is enteropathogenic but could spread to the bloodstream from the gut and disseminate to extra-intestinal organs. These results will contribute to our understanding of PSV pathogenesis.

Parasitological study and changes in level of some serum enzymes in Kano brown goats experimentally infected with <i>Trypanosoma evansi</i>

Trypanosomosis is a vector borne protozoan disease that is caused by varied members of the genus, Trypanosoma which affect different species of domestic and wild animals including humans. The disease processprovokes alterations in serum enzymes activities in the affected host. This study aimed at investigating the parasitological effects and changes in levels of some serum enzymes activities in Kano brown goats following experimental infection with T. evansi originally isolated from naturally infected camels at Maigatari. Infection was through the jugular veins of the experimental subjects (3 infected and2uninfected controls) at dose rate of 2.0 x 106/ ml. The period of study spanned 145 days. Sequel to the infection, infected goats developed clinical trypanosomosis and parasites were detected in the peripheral blood of all infected animals at varied prepatent period between 3 and 7 days post infection (DPI). First peak of mean parasitaemia wave was at 9 DPI and second at 14 DPI and death was recorded on the 70 and 133 DPI respectively among the infected group. After 84 DPI, no parasite could be detected and control subjects remained aparasitaemic throughout the experimental period. For serum enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), there was significant (P < 0.05) elevation in the infected subjects. The study showedT. evansi isolate caused infection caused changes in the activities of serum enzymes under study.Key words: Experimental infection, Kano brown goats, serum enzymes, Trypanosomosis,Trypanosoma evansi.

Correlations between the Levels of the Main Extracellular Matrix Components in Mouse Liver in Chronic BCG-Induced Granulomatosis.

Correlations between extracellular matrix components in mouse liver revealed the pathogenetically determined dynamic structure of these interrelations (some correlations appeared while others disappeared) at various terms of BCG-induced granulomatosis. The correlations between the studied parameters were strong during the first (postinfection days 3-10) and the second (postinfection days 60-90) periods but became moderate during the third period manifested by chronic inflammation (postinjection day 180). The greatest number of correlations was observed at the beginning of the stabilization period (postinjection day 60). At this period, the content of all structural components of hepatic proteoglycans closely correlated with that of hydroxyproline. These findings attested to systemic changes in metabolism of extracellular matrix components and interrelation between some structural units of proteoglycans in the liver and the components of extracellular matrix in the lungs. The common and typical organ-specific correlations in the liver and lungs suggest the existence of correlations between different organs during progression of BCG-induced granulomatosis.

Transcriptional Profile of Aedes aegypti Leucine-Rich Repeat Proteins in Response to Zika and Chikungunya Viruses.

Aedes aegypti (L.) is the primary vector of chikungunya, dengue, yellow fever, and Zika viruses. The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in invertebrates and vertebrates, as well as plants. We focused on the AaeLRIM1 and AaeAPL1 gene expressions in response to Zika virus (ZIKV) and chikungunya virus (CHIKV) infection using a time course study, as well as the developmental expressions in the eggs, larvae, pupae, and adults. RNA-seq analysis data provided 60 leucine-rich repeat related transcriptions in Ae. aegypti in response to Zika virus (Accession number: GSE118858, accessed on: August 22, 2018, GEO DataSets). RNA-seq analysis data showed that AaeLRIM1 (AAEL012086-RA) and AaeAPL1 (AAEL009520-RA) were significantly upregulated 2.5 and 3-fold during infection by ZIKV 7-days post infection (dpi) of an Ae. aegypti Key West strain compared to an Orlando strain. The qPCR data showed that LRR-containing proteins related genes, AaeLRIM1 and AaeAPL1, and five paralogues were expressed 100-fold lower than other nuclear genes, such as defensin, during all developmental stages examined. Together, these data provide insights into the transcription profiles of LRR proteins of Ae. aegypti during its development and in response to infection with emergent arboviruses.

Immune response against subclinical haemonchosis in Himalayan hill goats

Haemonchosis commonly occurs as chronic and subclinical infection in small ruminants, and understanding of immunological response against subclinical haemonchosis is of paramount importance for designing and implementing effective control strategies. The present study was designed to evaluate immunological response during subclinical haemonchosis, experimentally established in goats. Sixteen 5–6 month-old helminth naive kids were randomly allocated into one of two groups, infected and uninfected; the infected group being infected per os with 250 Haemonchus contortus larvae per kg body weight. Faecal, blood and serum samples were collected every third day up to 30 days post-infection (DPI), thereafter weekly up to 58 DPI to record changes in faecal egg count (FEC), haemoglobin (Hb), packed cell volume (PCV), peripheral eosinophil percentage and immunological parameters, such as macrophage cytokine interleukin-12 (IL-12), Th1 cytokine (IFN-γ), Th2 cytokines (IL-4, 13, 25, 33) and immunoglobulins (IgG and IgE). Pre-patent period of H. contortus in the present study was 18 days and eggs per gram (EPG) peaked on 30 DPI. The total reduction in body weight gain in the infected group was 26 g per day when compared with uninfected animals. Hb (7.35 ± 0.34 g/dL in infected animals compared with 9.76 ± 0.67 in control animals) and PCV levels (22 ± 1.54 g/dL in infected animals compared with 29.2 ± 1.27 in control animals) decreased significantly up to 44 DPI in infected group (P = 0.000). IL-4, IL-13, IL-33, IgG and IgE showed significant increase in infected animals at different periods. IFN-γ, IL-12 and IL-25 did not show any significant changes barring a steep rise of IFN-γ on 27 DPI. A positive correlation was observed between IgE and IL-4 in subclinical haemonchosis. Of particular note was that all the major cytokines, such as IFN-γ (P = 0.000), IL-4 (P = 0.000), IL-13 (P = 0.009), and both IgG (P = 0.000) and IgE (P = 0.003), were observed at the lowest concentration on 24 DPI. The effect of infection was found to be significant on cytokines with a strong interaction with time. Taken together, the data suggest that Th2 immune response is predominating in subclinical haemonchosis. The economic loss in term of body weight gain due to subclinical haemonchosis was considerable.

Biochemical Changes Caused by Eimeria spp in Broiler Chickens

This experimental study was performed to investigate the effect of coccidia infestation on biochemical parameters in broiler chicken. The experimental animals (n=100) were randomly allocated into four equal groups, group one (G-I), group two (G-II), group three (G-III) and group four (G-IV) with 25 chickens in each group. The G-I, G-II, and G-III were treatments groups challenged by different Eimeria sporulated oocysts, while G-IV served as the control group. In this study, the infective dose of E. tenella (G-I), E. acervulina(G-II) and mixed Eimeria spp (G-III)was 2×104 sporulated Eimeria oocyst inoculated orally at three weeks of age in broiler chicken and subsequent alterations in different plasma biochemical constituents were evaluated at interval of 5 , 7 and 9 day of post inoculation. Serum total protein values after challenge showed statistically significant decrease in group one, group two and group three in comparison with group four. Further, significant decrease total protien value was noticed on 7 day of post infection in group one and group two.The mean values of serum glucose between the infected and control group at 5, 7 and 9 day of post infection which revealed non-statistically significant difference. Coccidiosis due to E. tenella, E. acervulina and mixed identified Eimeria spp. infectionin chicken showed highly statistically significant increase in serum ALT and AST level as compared with control group. This was also significant increase in infected group on 7 day of post infection. But, no significant variation among the infected groups were on 5 and 9 day of post infection.

Persistence of Brucella abortus in the Bone Marrow of Infected Mice.

Brucellosis is a zoonotic bacterial infection that may persist for long periods causing relapses in antibiotic-treated patients. The ability of Brucella to develop chronic infections is linked to their capacity to invade and replicate within the mononuclear phagocyte system, including the bone marrow (BM). Persistence of Brucella in the BM has been associated with hematological complications such as neutropenia, thrombocytopenia, anemia, and pancytopenia in human patients. In the mouse model, we observed that the number of Brucella abortus in the BM remained constant for up to 168 days of postinfection. This persistence was associated with histopathological changes, accompanied by augmented numbers of BM myeloid GMP progenitors, PMNs, and CD4+ lymphocytes during the acute phase (eight days) of the infection in the BM. Monocytes, PMNs, and GMP cells were identified as the cells harboring Brucella in the BM. We propose that the BM is an essential niche for the bacterium to establish long-lasting infections and that infected PMNs may serve as vehicles for dispersion of Brucella organisms, following the Trojan horse hypothesis. Monocytes are solid candidates for Brucella reservoirs in the BM.

Safety and efficacy of attenuated classic and variant 2 infectious bronchitis virus candidate vaccines

ABSTRACTVaccination programs against infectious bronchitis virus (IBV) in Egypt depend on both classical and/or imported variant IBV strain vaccines. However, many IBV outbreaks associated with respiratory distress, nephropathy, and high mortalities were attributed to the circulation of both classical and new nephropathogenic IBV variant 2 strains. In the present study, we report the development of attenuated IBV candidate vaccines using the classic IBV strains (IBM41 and IB2) and a nephropathogenic strain (IBvar2). The wild-type (WT) viruses were attenuated through serial passages in embryonated specific pathogen free (SPF) chicken eggs. Virulence of the attenuated viruses was then tested via the ocular route inoculation and the in vivo back passage in day-old SPF chickens. Efficacy against homologous challenge was investigated also in day-old SPF chickens. Results showed that the viruses were successfully adapted to the embryo by the 100th (IBM41 and IB2) and 110th passages (IBvar2). The attenuated viruses were safe and showed no change of virulence in day-old SPF chickens up to the 10th back passages. The efficacy experiment showed that the attenuated vaccines showed 90 to 100% protection against the homologous challenge based on ciliostasis score and protection percent. The att-IBM41 and att-IB2 vaccines were able to reduce the shedding of the challenge at 3 days post-infection (DPI) and no virus shedding was detected in both vaccinated groups by 5 DPI. In the att-IBvar2 vaccinated birds, only 20% of vaccinated birds shed the challenge virus with low titers (102.10±0.3 EID50/mL) at 3 DPI. In conclusion, the attenuated strains IBM41, IB2, and IBvar2 are efficient vaccine candidates against currently circulating classic and variant IB viruses, respectively. Further studies to evaluate the field efficacy and combining these attenuated IBV strains to induce a wider protection against heterologous IBV challenge are suggested.

A Ciliary Motility Index for Activity Measurement in Cell Cultures With Respiratory Syncytial Virus.

The respiratory epithelium is frequently infected by the respiratory syncytial virus, resulting in inflammation, a reduction in cilia activity and an increase in the production of mucus. In this study, an automatic method has been proposed to characterize the ciliary motility from cell cultures by means of a motility index using a dense optical flow algorithm. This method allows us to determine the ciliary beat frequency (CBF) together with a ciliary motility index of the cells in the cultures. The object of this analysis is to automatically distinguish between normal and infected cells in a culture. The method was applied in 2 stages. It was concluded from the first stage that the CBF is not a good enough indicator to discriminate between the control and infected cultures. However, the ciliary motility index does succeed in discriminating between the control and infected cultures using the t test with a value t = 6.46 and P < .001. In the second stage, it has been shown that the ciliary motility index did not differ significantly between patients, and the analysis of variance test gives α = 0.05, F = 1.61, P = .20. A threshold for this index has been determined using a receiver operating characteristics analysis that gives an area under the curve of 0.93. We have obtained a ciliary motility index that is able to discriminate between control and infected cultures after the eighth postinfection day. After infection, there is a rapid cilia loss of the cells and the measured CBF corresponds to the remaining noninfected cells. This is why the CBF does not discriminate between the control and the infected cells.

Prostaglandin D2 Levels Regulate CD103+ Conventional Dendritic Cell Activation in Neonates During Respiratory Viral Infection.

During respiratory viral infection, conventional dendritic cells (cDCs) take up antigen and migrate to the draining lymph nodes to present viral antigen and activate cytotoxic T lymphocytes; however, regulation of cDC activation and migration may be age dependent. In this study, we used a mouse model of paramyxoviral infection (Sendai virus) and demonstrated that cDCs, which have migrated from lungs to the draining lymph nodes, are delayed in expressing activation markers in neonatal mice compared with adults. Neonatal lung cDCs expressed reduced levels of MHC Class II (major histocompatibility complex II) and CCR7 (chemokine receptor type 7) on postinfection days 3 and 5, respectively. The level of the CCR7 ligand CCL19 was significantly reduced in neonatal lungs during the course of viral infection. Interestingly, the arachidonic acid metabolite prostaglandin D2 (PGD2) was present at significantly higher levels in neonatal bronchoalveolar lavage fluid compared with adults. This was associated with increased expression of lipocalin PGD2 synthase mRNA levels in neonatal lungs and in isolated neonatal tracheal epithelial cells. Although thymic stromal lymphopoietin (TSLP) expression has been associated with increased PGD2 production, we found that TSLP levels were reduced in neonatal lungs. Importantly, blocking PGD2 function using a prostaglandin D2 receptor 1 (DP1) antagonist restored cDC activation in neonates. Together, these data suggest that cDC activation in neonates is delayed by a PGD2 mechanism and associated decreased chemokine signals.