Phylogenetic analysis of pathogenic Candida spp. in domestic pigeons.

The current study was conducted to survey the prevalence of pigeon candidiasis in diseased pigeons suspected to candidiasis by isolation, microscopic examination, and polymerase chain reaction (PCR) method and to characterize Candida spp. phylogenetically. For this purpose, samples were obtained from 100 suspected pigeons from September 2018 to February 2019 in Ahvaz, Iran. Cloacal and oropharyngeal swab samples were collected from each diseased pigeon with diarrhea resistant to the antibiotics, crop stasis, white diphtheritic membrane in the mouth, regurgitation, and vomiting. Sabouraud dextrose agar was used as a culture medium. Selected colonies were stained with lactophenol cotton blue stain. In the culture and direct microscopic observation, 19.00% of birds were suspected to candidiasis. Twenty-two isolates were identified. All 22 isolates were confirmed as Candida spp. By PCR method. The PCR test confirmed the presence of Candida spp. in 19.00% of pigeons. Based on the sequencing results of some PCR products, the isolates belonged to Candida albicans and Candida glabrata. The results revealed a 99.78% accordance when compared with other sequences of C. albicans which were formerly deposited in GenBank® from Colombia, Indonesia, China, and Sudan. The results revealed a 99.54% accordance when compared with other sequences of C. glabrata which were formerly deposited in GenBank® from the Netherlands and Spain. The symptoms such as diarrhea resistant to antibiotics, crop stasis, white diphtheritic membrane in the mouth, regurgitation, and vomiting were the most prevalent clinical symptoms in positive pigeons.

Avian gastric yeast (macrorhabdosis) in cockatiel, budgerigar and grey parrot: a focus on the clinical signs, molecular detection and phylogenetic evaluation.

Macrorhabdus ornithogaster is a microorganism that causes nonspecific and general clinical symptoms and to this day, diagnosis and also treatment have been yet hard. The present study was conducted to survey the prevalence of macrorhabdosis and to characterize M. ornithogaster phylogenetically in Psittaciformes suspected of macrorhabdosis from January 2018 to May 2019 in Ahvaz, Iran. For this purpose, fecal samples were collected from Psittaciformes with signs of the disease. Wet mounts were prepared from fecal samples and examined carefully using a light microscope. Samples from parrots with gastrointestinal symptoms of the disease were chosen for molecular diagnosis of the organism and DNA was extracted from these samples. For detection of M. ornithogaster, primer sets (BIG1, Sm4) and (AGY1, Sm4) which target the 18S rDNA gene were selected and Semi-nested polymerase chain reaction (Semi-nested PCR) was performed. The PCR method confirmed the presence of M. ornithogaster in 14.00% of the samples. Purified PCR products were sequenced for more accurate confirmation and according to the gene sequence all sequences were owned by M. ornithogaster. The results disclosed a 96.03 - 100% identity when compared to other sequences of M. ornithogaster which had previously been deposited in the GenBank® from Germany and the USA. The results of this study proved the circulation of M. ornithogaster between cockatiel, budgerigar and grey parrot. The prevalence of macrorhabdosis was higher in cockatiel compared to budgerigar and grey parrot. As far as the authors know, this was the first record of macrorhabdosis in African grey parrots.

Evaluation of hematological changes, oxidant/antioxidant status and immuno-logical responses in sheep and goats naturally infected with Linguatula serrata.

Linguatula serrata is a worldwide zoonotic food-borne parasite. The parasite is responsible for linguatulosis and poses a concern to human and animal health in endemic regions. This study investigated the hematological changes, oxidant/antioxidant status and immunological responses in goats and sheep naturally infected with L. serrata. Hematological changes, antioxidant enzymes and malondialdehyde (MDA) levels were measured. The level of inter-leukin-2 (IL-2), IL-4, IL-5, IL-10, and tumor necrosis factor alpha (TNF-α) mRNA expression was investigated in lymph nodes. According to the hemogram results, eosinophils were significantly increased in the infected goats and sheep, and Horizontal Gene Transfer (HGT), hematocrit (HCT), and mean corpuscular hemoglobin concentration (MCHC) were significantly decreased. The levels of MDA and the activity of glutathione peroxidase (GPx) were significantly higher in infected animals than in non-infected animals. However, the activity of superoxide dismutase (SOD) and catalase (CAT) was significantly lower in infected animals than in non-infected animals. A comparison of the cytokine mRNA expression in lymph nodes from infected and non-infected animals showed higher cytokine expression in the infected animals. Infection with L. serrata caused microcytic hypochromic and normocytic hypochromic anemia in goats and sheep. The inconsistent results of immunological changes were found in infected goats and sheep. In both animals, oxidative stress occurred and led to an increase in lipid peroxidation. L. serrata created a cytokine microenvironment biased towards the type 2 immune responses.

Determination of the fate of Cholecalciferol injected by the basis of 25D3 plasma concentration.

Sun exposure in bovines is believed to be the most important route of 25D3 synthesis in suitable latitudes. In some situations, e.g. breeding systems, solar radiation cannot reach or penetrate into the skin and thus causes the 25D3 deficiency. Because of the critical effect of vitamin D on the immune and endocrine systems, the plasma must be enriched with 25D3 in a short period of time. In such a condition, injection of Cholecalciferol has been recommended. However, to our knowledge, the certain dose of Cholecalciferol injection for rapid 25D3 plasma enrichment has not been verified. On the other hand, it seems that the basis 25D3 concentration can influence or shift the 25D3 metabolism at the injection time. In the same line, the present study, designed to induce the different basis 25D3 concentration in treatment groups, aimed at investigating the effect of Cholecalciferol intramuscularly injection with the intermediate dose (11,000 IU/kg) on the calves' plasma 25D3 with different basis 25D3. Besides, an attempt was made to clarify the time that 25D3 reaches the sufficient concentration after injection in different treatment groups. To do this, twenty calves of 3 to 4 months old were chosen for the farm with semi-industrial elements. Furthermore, the effect of optional sun exposure/deprivation and Cholecalciferol injection on the 25D3 concentration variations was assayed. To do this, the calves were divided into four groups. Groups A and B were unconstrained to choose sun to expose or shadow in a semi-roofed place, but groups C and D were restricted to the completely dark barn. The interference of the digestive system in supplying vitamin D was minimized through dietary. All groups had a different basic concentration (25D3) on the day 21 of the experiment. At this time, groups A and C received the intermediate dose of (11,000 IU/kg) Cholecalciferol intramuscularly (IM). After Cholecalciferol injection, the effects of basis 25D3 concentration on the details of variation and fate of plasma concentration of 25D3 were investigated. The data collected from the two groups C and D showed that sun deprivation without any vitamin D supplementation, could rapidly and severely deplete the plasma from 25D3. Cholecalciferol injection could not immediately increase the 25D3 in the groups C and A. However, this injection enriches the 25D3 to sufficient value after two weeks if the basis 25D3 of plasma is insufficient, i.e. less than 30 ng/mL. Moreover, the injection of Cholecalciferol could not significantly increase the 25D3 concentration in the group A that had a sufficient basis 25D3 concentration. Therefore, it is concluded that the variation of 25D3 in plasma, after injection of Cholecalciferol, depends on its basic level at the time of injection.

The role of small ruminants in the epidemiology of leptospirosis.

Leptospirosis is a common global zoonotic disease of man and all farm animals. Although most leptospiral infections in sheep and goats are asymptomatic, they may play a role in the epidemiology of the disease by the spread of Leptospira through the urine. This study was carried out to evaluate the role of sheep and goats in the epidemiology of leptospirosis. Blood and urine samples were taken from 210 goats and 246 sheep. To detect antibodies, sera samples were tested with 8 live serovars of L. interrogans (Hardjo, Pomona, Grippotyphosa, Canicola, Ballum, Icterhemorrhagiae, Tarasovi, and Australis) by MAT. Then, urine samples were tested by Nested PCR targeting 16S rRNA gene for detection of pathogenic Leptospira. Results of MAT showed that 10.95% of goats and 8.53% of sheep had antibodies against at least one examined serovars. In both species, the highest reacting was L. i. Pomona with a rate of 68.18% and 56% in sheep and goats, respectively. Moreover, in PCR, 2 (0.95%) urine samples of goat and 12 (4.87%) urine samples of sheep were positive. All of the MAT positive studied animals were PCR negative and, statistical analysis showed that there was no relationship and agreement between the results of PCR and MAT in sheep (kappa = - 0.07, p > 0.05) and goats (kappa = - 0.02, p > 0.05). Finally, it is concluded that sheep and goats can excrete L. interrogans in the urine and thus transmit them to other animals and humans.

Seroprevalence and risk factors of brucellosis in Arabian horses.

BACKGROUND: Brucellosis, as a zoonotic disease, mainly occurs in horses by Brucella abortus, Brucella canis and Brucella suis. The disease in equines is often asymptomatic, but the clinical signs in horses are mostly characterized by bursitis, arthritis and tenosynovitis. OBJECTIVES: This study, thus, aimed to determine the seroprevalence of brucellosis and its associated risk factors in the Arabian horses of Khuzestan province, South-west Iran. METHODS: To that end, the blood samples randomly collected from 180 Arabian horses were analyzed for the presence of anti-Brucella antibodies by Rose Bengal plate test (RBPT), serum agglutination test (SAT), 2-mercaptoethanol test (2-ME) and a commercial i-ELISA kit. RESULTS: The ROC curve analysis showed that the best cut-off point for S/P values in i-ELISA turned out to be 26.25%. The results showed that the overall seroprevalence of brucellosis based on parallel interpretation of the test results was 12.22% (Positive/Tested = 22/180). The prevalence of acute and chronic brucellosis was 8.3 and 3.9%, respectively. The seroprevalence of brucellosis with RBPT and i-ELISA methods was 1.11% (2/180) and 7.22% (13/180), respectively. According to what SAT revealed, 9.44% (17/180) of sera had a titer of 40 or greater, and at 2-ME, 7.22% of samples (13 out of 180 samples) depicted a titer of 40. The results of i-ELISA, SAT and 2-ME were significantly different from those of RBPT (p < 0.01); however, there was no significant difference between i-ELISA, SAT and 2-ME in findings (p > 0.05). CONCLUSIONS: The results of this study recommend that i-ELISA be used for screening purposes of brucellosis in horses. The findings confirmed that Arabian horses are natural hosts for the Brucellae. It is, thus, necessary to adopt appropriate prevention and control programs by health authorities and horse owners so as to reduce the distribution and transmission of the infection in the regions where brucellosis is prevalent.

Genotyping of Chlamydia abortus using multiple loci variable number of tandem repeats analysis technique.

BACKGROUND: The correlation between various factors (geographical region, clinical incidence, and host type) and the genomic heterogeneity has been shown in several bacterial strains including Chlamydia abortus. METHODS: The aim of this study was to survey the predominant types of C. abortus strains isolated from ruminants in Iran by the multiple loci variable number of tandem repeats (VNTR) analysis (MLVA) method. C. abortus infection was evaluated in a total of 117 aborted fetuses by real-time PCR. The isolation was done via the inoculation of the positive samples in chicken embryo and the L929 cell line. Genotyping was carried out by MLVA typing technique. RESULTS: Forty samples (34.2%) were detected with C. abortus infection; however, chlamydial infection in ruminants of Charmahal/Bakhtiari (3 bovines and 35 sheep) was higher than that of Khuzestan (2 sheep). All MLVA types (MT1-MT8) were detected in the collected samples from Charmahal/Bakhtiari but only 2 types (MT1 and MT3) were reported in samples from Khuzestan. The main MT type was MT1 (32% of aborted fetuses). Although in this study only 9 cow samples were investigated, they possessed similar clusters to those obtained from sheep (MT1 and MT6). Variation of type in sheep samples (MT1 to MT8) was more than that of bovine samples (MT1, and MT6). CONCLUSION: By this research revealed that C.abortus was responsible for a significant percentage of ruminant abortion in two studied regions. The main MT type was MT1 (32% of aborted fetuses) and also 7 different genotypes were involved in infections. So it is concluded that diversity in C.abortus genotyping is high in two regions.

Candidiasis in Birds (Galliformes, Anseriformes, Psittaciformes, Passeriformes, and Columbiformes): A Focus on Antifungal Susceptibility Pattern of Candida albicans and Non-albicans Isolates in Avian Clinical Specimens.

Candidiasis is a fungal infection caused by Candida species which has been reported in most domestic and wild birds and mammals. In this study, 196 samples from different species of birds with suspected symptoms of candidiasis were examined. Pharyngeal swabs, cloacal swabs, and fecal samples were taken from the birds. The samples were cultured in sabouraud dextrose agar (SDA) containing cycloheximide and chloramphenicol and incubated at 42°C. Suspected isolates of Candida were identified using PCR. To detect the candida genus, a primer set to target the candida rDNA (ITS1-ITS4) was selected. To detect Candida albicans (C albicans), a primer set to target cytochrome P-450 lanosterol-a-demethylase (P450-LIAl) gene (DH-1558) was selected. In direct microscopic observation and culture, 28.57% of the birds were suspected of candidiasis. In the molecular study, candidiasis was confirmed in 25% of the birds, and candidiasis caused by C albicans was confirmed in 14.28% of the birds. All isolates were subjected to antibiotic susceptibility by the disk diffusion method with glucose-enriched Mueller-Hinton Agar. 78.5% of the isolates were sensitive to nystatin and amphotericin B. None of the isolates were sensitive to itraconazole and more than 50% of the isolates were resistant to fluconazole, ketoconazole, and itraconazole. According to the results, it is suggested to use nystatin and amphotericin B in the treatment of avian candidiasis in the Ahvaz region. To the authors' knowledge, this is the first report of the molecular detection and antifungal susceptibility pattern of C albicans and non- albicans from Galliformes, Anseriformes, Psittaciformes, and Passeriformes in Iran.

An Enzyme-Linked Immunosorbent Assay for the Detection of Antibodies to Linguatula serrata in Experimentally Infected Dogs.

BACKGROUND: Linguatula serrata is a causative agent of visceral and nasopharyngeal linguatulosis in humans and animals. The aim of the present study was to investigate the immune response of dogs experimentally infected by L. serrata with ELISA. METHODS: Five puppies were infected by inserting the L. serrata nymphs in their nasal cavities (infected group) in the Department of Parasitology of Shahid Chamran University of Ahvaz, during 2018-2019. Three animals were kept as the non-infected control group. Blood samples were collected from the animals for seven months at approximately monthly intervals for serum preparation. Nasal samples were taken weekly from the fourth month. ELISA was designed and performed on 64 sera (24 negatives, and 40 positives) using somatic (S), and excretory-secretory (ES) antigens. RESULTS: Overall, 100% of the animals were infected with the parasite. Based on the results of ELISA, the ES antigen (sensitivity 95% and specificity 92%) was more preferred than the S antigen (sensitivity 95% and specificity 85%). Female parasites had significant effects on the immune response. There was a significant correlation between the clinical symptoms and the presence of female parasites (P<0.05). CONCLUSION: The results showed a practical method for dogs' experimental infection. ELISA method is suitable for the detection of infection at different stages of development, especially before the maturation stage of the parasite. In this regard, the ES antigen of the parasite was more immunogenic. Therefore, ELISA can be used as a serological method in the early detection and epidemiological studies of infection with L. serrata in dogs.

Comparison of Mycobacterium avium subsp. paratuberculosis infection in cattle, sheep and goats in the Khuzestan Province of Iran: Results of a preliminary survey.

BACKGROUND: Paratuberculosis or Johne's disease, the chronic infectious granulomatous enteritis of ruminants, is a worldwide infection, which is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The most common symptoms of this disease in cattle are loss of milk production, weight loss and diarrhoea, whereas in sheep and goats, the symptoms are emaciation, anorexia and severe disability. OBJECTIVES: The aim of this study was to compare the seroprevalence of MAP in cattle, sheep and goats in the southwest of Iran. METHODS: Blood samples were randomly collected from 530 cattle, 568 sheep and 368 goats in southwest of Iran. Sera were tested by a commercial ELISA kit (ID vet; ID Screen® Paratuberculosis Indirect) for detection of antibodies of MAP. RESULTS: Overall apparent and true seroprevalence rate of MAP was 6.00% (95% CI: 4.90%-7.30%) and 13.25% (95% CI: 11.55%- 14.95%). Apparent and true seroprevalence of MAP, respectively, was 4.34% (95% CI: 3.88%-6.46%) and 9.19% (95% CI: 6.98%-11.98%) in cattle, 6.87% (95% CI: 5.05%-9.27%) and 15.37% (95% CI: 12.60%-16.60%) in sheep and 7.07% (95% CI: 4.82%-10.18%) and 15.86% (95% CI: 12.41%-20.01%) in goats, respectively. As a result, there was no significant relationship between animal species and MAP infection. Moreover, multivariate logistic regression showed that the infection rate is not associated with age, gender and geographical location in cattle, sheep and goats (P > 0.05). CONCLUSION: This study confirms that the seroprevalence of MAP is relatively considerable in the cattle, sheep and goats in the southwest of Iran, although in cattle, it is less than goats and sheep. Therefore, preventive and control measures should be considered by animal health authorities and meat and dairy processing units.

Toxigenic and non-toxigenic Pasteurella multocida genotypes, based on capsular, LPS, and virulence profile typing, associated with pneumonic pasteurellosis in Iran.

Pasteurella multocida is an important cause of pneumonic pasteurellosis in small ruminants. Its prevalence was investigated in 349 pneumonic lungs from sheep (n = 197) and goats (n = 152), and genotypes of isolates were determined by capsular and lipopolysaccharide (LPS) typing as well as by virulotyping based on the detection of 12 virulence-associated genes. P. multocida was isolated from 29.4 % of sheep lungs and 13.8 % of goat lungs. A (78.5 %) and D (21.5 %) capsular types, as well as L3 (41.8 %) and L6 (57.0 %) LPS genotypes, were detected, with the A:L6 genotype being the most prevalent in both sheep (59.6 %) and goat (52.4 %) isolates. A total of 19 virulence profiles (VP) were detected, seven non-toxigenic and 12 toxigenic, which correlated with the capsular-LPS genotype. All isolates of each VP belonged to the same LPS and capsular genotype, except for one isolate of VP1. The diversity in VP was higher among toxigenic (0.29) than non-toxigenic (0.18) isolates. Moreover, the toxigenic VPs showed more diversity in their capsular-LPS genotypes, with the two main toxigenic VPs belonging to genotypes D:L3 (VP2) and A:L3 (VP3). Therefore, the abundance of toxigenic isolates among sheep and goat isolates does not seem to correspond to the expansion of a more virulent lineage associated with pneumonic pasteurellosis in small ruminants. The most prevalent genotypes among sheep isolates were the non-toxigenic VP1:A:L6 (41.4 %) and the toxigenic VP3:A:L3 (17.2 %) genotypes, whereas the most prevalent among goat isolates were the toxigenic VP2:D:L3 (33.3 %) and the non-toxigenic VP1:A:L6 (14.3 %) and VP4:A:L6 (14.3 %) genotypes. These prevalent toxigenic and non-toxigenic genotypes seem to be epidemiologically relevant in pneumonic pasteurellosis of small ruminants.

Isolation, molecular identification, and phylogenetic evaluation of Cryptococcus neoformans isolated from pigeon lofts, Psittaciformes, and Passeriformes in Ahvaz, Iran.

Cryptococcus neoformans, the main pathogen in immunocompromised patients, is a ubiquitous free-living fungus that can be isolated from avian excreta, soils, and plant material. This study was carried out to determine the infection rate of pigeon lofts, Passeriformes, and Psittaciformes in Ahvaz, the capital of Khuzestan province in Iran and to determine varieties of Cryptococcus neoformans (C. neoformans). The 80 samples were collected from pigeon lofts. Also, 163 feces of captive birds (Passeriformes and Psittaciformes) which kept in Ahvaz pet shops, and the 70 cloacal swabs of pet birds (Passeriformes and psittaciformes) referring to the department of avian medicine (the faculty of veterinary medicine of Shahid Chamran University of Ahvaz) were analyzed. The samples were directly inoculated on niger seed agar (NSA) and also enriched in brain heart infusion broth and then inoculated on NSA. Dark brown colonies suspected to C. neoformans subcultured on saborouds dextrose agar and pure cultures subjected to molecular (polymerase chain reaction (PCR)) diagnosis. For detection of C. neoformans, primer sets that targeting the CNLAC1 gene were selected and nested PCR was conducted. For identification of C. neoformans varieties, a primer set targeting the STR1 gene was selected. For more accurate confirmation, the purified PCR products of some isolates were also sequenced, and based on the gene sequences, all of the isolates belonged to C. neoformans variety grubii (var. grubii)(serotype A). Totally 16 out of 80 pigeon samples (20%) were contaminated with C. neoformans. The results in pigeons disclosed a 98.64% identity when compared with other strains of C. neoformans (CN1525, T4, and T1) which were previously deposited in GenBank from Italy and Thailand. Also, 21 out of 233 samples from Psittaciformes (9.01%) were contaminated with C. neoformans. The results in Psittaciformes disclosed a 99.7% identity when compared with other strains of C. neoformans (TIMM1313, IFM5882, CN1525, etc.) which were previously deposited in GenBank from Japan and Italy, etc. In the present study, the samples belonging to the passerine order were free of C. neoformans infection. According to the results, C. neoformans is prevalent in pigeon flocks and pet birds including Psittaciformes in the Ahvaz area, and should be considered by pigeon and captive bird breeders, veterinarians, and public health organizations in Ahvaz. The cryptococcus species isolated from captive birds and pigeons could be potential pathogens in humans.

Molecular characterization of Mannheimia haemolytica associated with ovine and caprine pneumonic lung lesions.

This study investigated via polymerase chain reaction (PCR) three main serotypes (A1, A2, and A6) and nine virulence-associated genes in 71 ovine and caprine Mannheimia haemolytica isolates obtained from lungs (n = 349) with pneumonic lesions from a slaughterhouse in Iran. The lung specimens were collected from sheep (n = 197) and goats (n = 152) between December 2018 and January 2020. A total of 71 M. haemolytica isolates were identified in sheep (37/197; 18.8%) and goat (34/152; 22.4%) pneumonic lungs. Serotypes A2 (30/71; 42.3%) and A6 (29/71; 40.9%) were the most frequently detected, whereas the A1 serotype was detected with a frequency of less than 10% (7/71; 9.9%) and five isolates remained unknown. The virulence genes lkt, pomA, and nanH were present in all the isolates. The detection rates for the remaining virulence-associated genes were: gcp (95.8%), lpsA (93%), fhaC (90%), irp (70.4%), hf (57.7%), and sodC (21%). The sodC gene was exclusively detected among A2 isolates (50%), while the irp gene was more prevalent among A2 isolates and the hf gene among A1 and A6 isolates. These data may be useful for the typing of isolates in epidemiological studies. This study provides information about the main serotypes and the prevalence of virulence-associated genes among M. haemolytica ovine and caprine isolates in Iran.

The potential therapeutic effect of adipose-derived mesenchymal stem cells in the treatment of cutaneous leishmaniasis caused by L. major in BALB/c mice.

Leishmaniasis is one of the most neglected tropical infectious diseases in the world. The emergence of drug resistance and toxicity and the high cost of the available drugs with a lack of new anti-leishmanial drugs highlight the need to search for newer therapies with anti-leishmanial activities. Due to the mesenchymal stem cell (MSC) immunomodulatory capacity, they have been applied in a wide variety of disorders. In this study, the potential effects of adipose-derived MSC (AD-MSCs) therapy and its combination with glucantime were evaluated in a murine model of cutaneous leishmaniasis induced by L. major. The results showed that AD-MSCs improved wound healing and decreased parasite burden. The real-time PCR results obtained from mice treated with AD-MSCs showed that IL-12 and TNF-α genes were upregulated. IL-10, arginase, and FOXP3 genes were downregulated whereas no differences in expression of the IL-4 gene were found. Overall, it seems that AD-MSCs therapy enhances Th1 immune response in L. major infected BALB/c mice. Unexpectedly, our results showed that the association of glucantime to AD-MSCs treatments did not lead to an increment in the anti-leishmanial activity.

Immunogenicity and protective efficacy of Yersinia ruckeri lipopolysaccharide (LPS), encapsulated by alginate-chitosan micro/nanoparticles in rainbow trout (Oncorhyncus mykiss).

Considering the many advantages of oral vaccines in aquaculture, several studies have been conducted in this area recently. In this study, immunization and protective power of the oral vaccine of Yersinia ruckeri encapsulated with Alginate-Chitosan micro/nanoparticles were evaluated in rainbow trout. For this purpose, 720 juvenile rainbow trout (9 ± 1.8 g) were divided into 8 groups in three replications (30 fish each) as follows: Groups A, B and C, were immunized with Yersinia ruckeri lipopolysaccharide (LPS), LPS+Formalin Killed Cells (FKC) and FKC alone, groups D, E, and F were immunized with encapsulated LPS, LPS+FKC and FKC, respectively. The G and H groups considered as encapsulated and non-encapsulated control, respectively. Micro/nanoencapsulation with alginate-chitosan was performed by internal emulsification method and vaccination were conductrd in the first and third weeks via oral route. Sampling was performed on days 0, 30, and 60 of experiment. Anti Y. ruckeri antibody titer in serum, intestine and skin mucus were measured via ELISA method. Non-specific immune response including: serum lysozyme, complement, bactericidal and respiratory burst activity, serum protein and globulin level, as well as white blood cell count were compared among the groups. The expression of IgT gene in the intestine and TCR gene in the anterior kidney were also investigated. At the end of the study, the fish were challenged with Y. ruckeri through immerssion and intraperitoneal routs and the relative survival rate was evaluated. Result showed that the antibody level in serum, skin and intestine was significantly higher in group E and F than control groups (P < 0.05), meanwhile serum, skin and intestine antibody level in all vaccinated groups were significantly (P < 0.01) higher in day 30 and 60 compare to zero day. Non-specific immunity factors including: serum lysozyme, complement, and respiratory burst activity as well as WBC, protein and Globulin level were significantly higher in E and F groups not only in day 30 but also in day 60 of experiment (P < 0.05). Cumulative mortality following injection and bath challenge were significantly (P = 0.004) lower (35%-45%) in groups E and F compare to control group (80%). The IgT and TCR gene expression in groups D, E and F were significantly higher (P < 0.05) than control group. Highest upregulation of IgT and TCR gene expression in vaccinated groups were seen at day 30 and 60 respectively which were significantly (P < 0.001) higher than day zero. Generally, it can be concluded that nano/micronanoencapsulation of Y. ruckeri FKC+LPS with chitosan-alginate, not only increases protective efficacy of oral vaccine, but improves specific and non-specific immune responses in rainbow trout.

High efficacy and economical procedure of oral vaccination against Lactococcus garvieae/Streptococcus iniae in rainbow trout (Oncorhynchus mykiss).

The present study was aimed to examine the efficacy of chitosan-alginate coated vaccines against pathogenicity of Lactococcus garvieae and Streptococcus iniae in rainbow trout. Fish were divided into four groups including: Group A: fish immunized by chitosan-alginate coated vaccine, Group B: fish immunized by non-coated vaccine, Group C: fish feed by chitosan-alginate coated pellets without vaccine and Group D: fish feed by basic diet (non-coated and without vaccine). In groups A and B, the vaccination was carried out for 14 days and after that supplemented with fundamental diet (control diet). Comparable to groups A and B, fish of group C were also fed 14 days with test diets and after that fed control food. On day 0, 20, 40 and 60 of the experiment, serum samples were given. Fish have been challenged with live L. garvieae and S. iniae after 60 days. The levels of bactericidal activity and complement activity among innate immunity components extended on day 20 of the research and after that decreased in group A and B (P < 0.05) all through the examination. The relative expression of IL-6 and IgM in groups A and B extended on examination day 20. The expression of these genes illustrated no advancements in different groups in during the examination (P > 0.05). In group A, the serum antibody titer against L. garvieae and S. iniae broadly raised on day 40 and 60 of examination, whereas in group B, the immune response titer against S. iniae and L. garvieae illustrated a significant elevation on day 60 of the trial (P < 0.05). After challenge with live bacteria, survival rate of 83 ± 9.1%(challenged with S. iniae) and 72.18 ± 9.8% (challenged with L. garvieae) were gotten independently in group A, which were higher than survival of other exploratory groups (P < 0.05). In conclusion, the results of the present examination appear that the orally vaccination of rainbow trout with chitosan-alginate covered vaccine stimulates immunity system and also efficiently protects rainbow trout against Lactococcus garvieae and Streptococcus iniae.

Detection of multi-antibiotic resistant Campylobacter coli and Campylobacter jejuni in beef, mutton, chicken and water buffalo meat in Ahvaz, Iran.

Campylobacter jejuni and C. coli are the main causes of gastrointestinal diseases in humans even in industrialized countries affecting public health. The aim of the current study was to evaluate the occurrence and antibiotic resistance of C. jejuni and C. coli in chicken meat, beef, mutton and water buffalo meat slaughtered in Ahvaz city, Iran. A total of 380 samples including chicken meat from industrial abattoirs (n = 150), chicken meat from traditional abattoirs (n = 50), fresh packed chicken meat from local markets (n = 30) and beef, mutton and water buffalo meat from industrial abattoirs (50 samples for each meat) in Ahvaz,were collected and tested for the presence of Campylobacter spp. The procedure was one-step enrichment in Preston enrichment broth followed by plating on supplemented blood agar for 24 hr under microaerophilic conditions at 42 ˚C. Suspected colonies were tested by polymerase chain reaction assay and susceptibility of the confirmed isolates to various antibiotics was investigated by the Kirby-Bauer disk diffusion method. Overall, 32 samples (8.40%) were contaminated with Campylobacter spp. Mutton was the most contaminated meat (24%), while fresh packed chicken meat were not contaminated. Among the 32 isolates, 40.60%, 34.40%, 21.90%, and 15.60% were resistant to tetracycline, ciprofloxacin, ampicillin, and streptomycin, respectively. Moreover, a high number of multi-antibiotic resistant Campylobacter spp. was determined. Since foods of animal origin are the most sources of Campylobacter infection, the presence of resistant strains to antibiotics is a potential risk to public health.

Valuable method for production of oral vaccine by using alginate and chitosan against Lactococcus garvieae/Streptococcus iniae in rainbow trout (Oncorhynchus mykiss).

The effectiveness of ionotropic gelation method (by combining alginate and chitosan) vaccine against Lactococcus garvieae and Streptococcus iniae was examined in rainbow trout. Fish were separated into four groups and fed the distinctive examined feeds. Our groups were included: A) fish immunized by chitosan-alginate coated vaccine, B) fish immunized by non-coated vaccine, C) fish feed by chitosan-alginate coated pellets without vaccine and D) fish feed by basic diet (non-coated and without vaccine). In groups A and B, the vaccination was carried out for 14 days. Fish of group C, like groups A and B were fed 14 days with pellets covered with chitosan-alginate without vaccine and a short time later they were fed with control diet. On day 0, 20, 40 and 60 of the trial, serum samples were extracted. Fish were challenged with L. garvieae and S. iniae after 60 days of research. Innate immunity components containing complement activity, total protein and IgM appeared no significant changes nearly in all groups during the 60 days that the examination finished. Although, bactericidal activity and lysozyme activity demonstrated a significant increase on days 20, 40 and 60 in group A compared to control groups (C and D) (P < 0.05) and similar results about the blood respiratory burst activity just on days 20 and 40 were obtained. Also, the relative expression of IL-6 of group A, was significantly higher compared to all of other groups (B, C and D) on days 20 and 60 of experiment (P < 0.05). The same results were obtained about the relative expression of IgM. The serum ELISA antibody titer against L. garvieae, increased significantly on days 20 and 40 of experiment in fish immunized by chitosan-alginate coated vaccine (Group A) compared to control groups (C and D)(P < 0.05) while the result of ELISA test against S. iniae was significantly higher on days 40 and 60 of experiment in group A compared to groups B, C and D (P < 0.05). After challenge with these two live bacteria (S. iniae and L. garvieae), a survival rates of 76.67 ±â€¯5.77% (challenged with S. iniae) and 66.67 ±â€¯5.77% (challenged with L. garvieae) were seen in group immunized with chitosan-alginate coated vaccine (Group A), which were higher than survival rates gotten in other trial groups (P < 0.05). The consequences of the present experiment show that the oral vaccination of rainbow trout with improved chitosan-alginate (via ionotropic procedure) (group A) properly secures this important fish against Lactococcus garvieae and Streptococcus iniae.

Effect of experimental infectious bursal disease virus on clinical signs and pathogenesis of avian influenza virus H9N2 in turkey by real time PCR.

Infectious bursal disease virus (IBDV) in turkeys may result in immunosuppression, and inability of turkeys to resist nonpathogenic or less pathogenic organisms. A total number of 120 day-old commercial male turkeys were purchased and blood samples were collected from 20 day-old turkeys, remaining 100 were divided into four equal groups and kept in separated rooms. Groups 1 and 2 were infected with 104 CID50 of IBDV via intra-bursal route on day 1; Groups 1 and 3 were each infected with 106 EID50 of AIV (H9N2) via the oculo-nasal routes on day 30. All groups were vaccinated against Newcastle disease vaccine (NDV). Detection of avian influenza virus H9N2 in trachea and cloaca swabs and in the tissues, was confirmed by Real-time polymerase chain reaction. Anti- NDV-AIV and anti-IBD titers were measured using HI and ELISA tests, respectively. The present study showed that infectious bursal disease changed the pathogenesis of (AIV) H9N2 by affecting AI virus replication and resulted in an increase shedding due to prolonged duration of sever clinical signs. The extent of shedding and virus replication need further study.

Efficacy of a Eudragit L30D-55 encapsulated oral vaccine containing inactivated bacteria (Lactococcus garvieae/Streptococcus iniae) in rainbow trout (Oncorhynchus mykiss).

The efficacy of a Eudragit L30D-55 encapsulated vaccine against Lactococcus garvieae and Streptococcus iniae was investigated in rainbow trout. Fish were divided into four groups and fed the different experimental feeds. Groups were: A) fish immunized by Eudragit-coated pellets containing vaccine, B) fish immunized by vaccine-coated pellets without Eudragit, C) fish fed Eudragit-coated pellets without vaccine and D) fish fed pellets without vaccine orEudragit (control group). In groups A and B, the vaccination was conducted for 14 days. Similar to groups A and B, fish of group C were fed 14 days with pellets coated with Eudragit and afterwards they were fed control diet. Serum samples were taken on day 0, 20, 40 and 60 of the experiment. After 60 days, fish were challenged with L. garvieae and S. iniae. In almost all groups, innate immunity components including alternative complement activity, lysozyme activity, bactericidal activity, IgM and total protein showed no significant changes during the 60 days that the experiment lasted. However, the blood respiratory burst activity and lysozyme activity showed a significant increase on day 20 of experiment in groups B and D respectively (P < 0.05). The relative expression of immune-related genes including IL-6 and IgM genes was higher in vaccinated fish, with the highest expression in those immunized by Eudragit-coated pellets (Group A). In addition, the relative expression of IL-6 and IgM peaked on day 20 but decreased on day 60 in vaccinated groups. The ELISA antibody titer against L. garvieae increased from day 20 and peaked on day 60 of experiment (P < 0.05). Also, the antibody titer against L. garvieae was higher in fish immunized by Eudragit-coated pellets (Group A) compared to fish of group C and control. After bacterial challenge, a survival percentages of % 85 ±â€¯7.07% (challenged with S. iniae) and % 72.21 ±â€¯7.8% (challenged with L. garvieae) were observed respectively in groups immunized with pellets coated with Eudragit L30D-55 (Group A), which were higher than survival percentages obtained in other experimental groups (P < 0.05). The results of the present study demonstrate that the oral administration of Eudragit L30D-55-encapsulated vaccine appropriately protects rainbow trout against Lactococcus garvieae and Streptococcus iniae.