Coxiella burnetii and Borrelia spp. in peripheral blood of dromedary camels in Fars, Iran: molecular characterization, hematological parameters, and acute-phase protein alterations.

Background: Dromedary camels (Camelus dromedarius) are raised in extremely strict ecological conditions of deserts. Camels are vulnerable to many zoonotic infections. There are limited data on the occurrence of Q fever and borreliosis in camels, in Iran. Aims: The current study was focused on the occurrence of Coxiella burnetii and Borrelia spp. infection in the blood samples of Iranian camels using molecular assays. Effect of the presence of these infections on various hematological factors and some acute-phase proteins (Hp, a1AGP, SAA) were also investigated. Methods: Blood samples were collected from 113 clinically healthy camels to investigate the presence of the infections using nested PCR. Moreover, the sequence of positive samples was analyzed phylogenetically. Routine haematological tests were performed and the concentrations of acute-phase proteins were measured in serum using enzyme immunoassay. Results: PCR result showed that 6.19% (95% CI: 2.53-12.35%) (7/113) of camels were positive for C. burnetii. In addition, sequencing results of the corresponding gene of the outer membrane protein (com1) revealed two different genotypes of C. burnetii agent in camels from Southern Iran. In the PCR assay, Borrelia spp. DNA was not detected in the samples. No significant difference was observed in hematological parameters or acute-phase proteins between positive and negative Q fever camels except for mean corpuscular hemoglobin (MCH) and red cell distribution width (RDW). Conclusion: Clinically healthy camels might be very important reservoirs of zoonotic pathogens. Q fever is not considered a notifiable disease in camels of Iran, and clinical cases may scarcely be recognized by the healthcare system. Due to a lack of adequate information, additional studies on the molecular epidemiology and clinical pathology aspects of C. burnetii infection in Iran are needed.

Effects of citral on serum antioxidant status and liver genes expressions of paraoxonase 1 and nitric oxide synthase in a rat model of streptozotocin-induced diabetes mellitus.

BACKGROUND: Citral (C10H16O) is the main ingredient of Cymbopogon citratus (lemongrass oil) and can reduce the side effects of oxidative stress. Diabetes caused by insulin deficiency induces oxidative stress in the liver. AIMS: This study aimed to investigate the ameliorative effects of citral on selected oxidative parameters and the gene expression of paraoxonase 1 (PON1) and endothelial nitric oxide synthase (eNOS) in a rat model of streptozotocin (STZ)-induced diabetes mellitus. METHODS: Forty rats were divided into four groups at random: control (C), control citral (CC), and two STZ-induced diabetic groups (diabetic (D) and citral diabetic (CD)). After diabetes confirmation (day 7), gavage treatment with citral (300 mg/kg body weight (BW)) was started in the CD and CC groups and continued for two weeks. RESULTS: On day 21 of the study, following treatment with citral for 14 days, the serum levels of total antioxidant capacity (TAC), and PON1 in the CD group were significantly increased compared to those in the D group (P<0.05). While treatment with citral caused a significant decrease in the Malondialdehyde (MDA), and eNOS in the CD group compared to those of the D group (P<0.001). The expression rate of liver PON1 gene was considerably upregulated in the CD group compared to that in the D group (P<0.001); while the opposite was observed for eNOS gene expression. However, there was no significant difference between the CC and C groups in terms of all examined parameters (P>0.05). CONCLUSION: This study showed positive effects of citral on serum antioxidant status and liver gene expression of PON1 and eNOS in diabetic rats.

Serotypes, virulence genes and polymorphism of capsule gene cluster in Lactococcus garvieae isolated from diseased rainbow trout (Oncorhynchus mykiss) and mugger crocodile (Crocodylus palustris) in Iran.

BACKGROUND: Lactococcus garvieae causes lactococcosis in rainbow trout in many parts of the world. AIMS: This study was conducted for the existence of the virulent factors and differentiation of the two serotypes in L. garvieae. METHODS: Twenty-two strains of L. garvieae isolated from diseased rainbow trout from farms in different regions and mugger crocodile of Iran, were investigated. In order to rapidly detect the presence of the hly1, hly2, hly3, NADH oxidase, sod, pgm, adhPsaA, eno, LPxTG-3, adhCI, and adhCII virulence genes, two multiplex polymerase chain reaction (PCR) assays were developed. Also, simplex PCR method was used to identify the bacterial serotypes, CGC, LPxTG-2, Adhesion, and adhPav virulence genes using the specific primer. RESULTS: All varieties of L. garvieae contained the hly1, hly2, hly3, NADH oxidase, pgm, adhPav, LPxTG-3, sod, eno, adhPsaA, adhCI, and CGC virulence genes. Also, adhCII gene was present in all strains except one of the isolates originated from mugger crocodile. In addition, LPxTG-2 gene was only present in one of the isolates belonging to mugger crocodile. Adhesion gene was not present in all the strains. Interestingly, all the 22 strains originated from both hosts were identified as belonging to the serotype I. Based on the phylogenetic sequences of the capsule gene cluster, group all fish isolates into a cluster together with one isolate obtained from mugger crocodile. CONCLUSION: Further studies are recommended to investigate the role of virulence genes in L. garvieae and evaluate their pathogenicity to rainbow trout.

Molecular and clinical evidence of Aeromonas hydrophila and Fusarium solani co-infection in narrow-clawed crayfish Astacus leptodactylus.

Co-infections occur when a host is infected by 2 or more different pathogen types, either by secondary or simultaneous infections, and are very frequent in nature. In this study, 10 narrow-clawed crayfish Astacus leptodactylus with signs of disease were collected from Haft Baram Lake (Fars province, southern Iran). Samples of fluid from inside the intact abscess and melanized lesions in the cuticle were cultured aseptically onto brain heart infusion agar and Sabouraud dextrose agar for bacterial and fungal agents, respectively. After primary colony isolation for bacterial and fungal agents, the isolates were confirmed as Aeromonas hydrophila and Fusarium solani, using specific PCR methods based on 16S rDNA and internal transcribed spacer (ITS) rDNA sequences that produced a single band of 685 bp and 600 bp, respectively. Partial sequence analysis of the F. solani ITS showed 100% sequence identity among all our samples, as well as a close genetic relationship between this isolate (GenBank accession number MG519784) and those previously reported from loggerhead sea turtle Caretta caretta in Cape Verde (FJ948133, AM412641, and DQ535186), black-blotched stingray Taeniura melanopsila in Japan (LC019016), and American manatee Trichechus manatus in Japan (AB775569). The results indicate that narrow-clawed crayfish can be infected by A. hydrophila and F. solani simultaneously, and to the best of our knowledge, this is the first report of just such a co-infection in this host. Further studies are necessary to investigate the pathogenicity of these organisms and their effects on narrow-clawed crayfish.

Identification of N-acyl homoserine lactone-degrading bacteria isolated from rainbow trout (Oncorhynchus mykiss).

AIMS: A variety of pathogens use quorum sensing (QS) to control the expression of their virulence factors. QS interference has hence been proposed as a promising antivirulence strategy. The specific aim of this study was to isolate bacteria from trout tissue able to degrade N-acyl homoserine lactones (AHL), a QS molecule family. METHODS AND RESULTS: In total 132 isolates were screened for AHL degradation using Chromobacterium violaceum CV026 as a biosensor. Twenty-four quorum-quenching (QQ) isolates were identified biochemically and characterized using 16S rDNA sequencing. They belong to Bacillus, Enterobacter, Citrobacter, Acinetobacter, Agrobacterium, Pseudomonas and Stentrophomonas genera. Four Bacillus spp. showed the highest and fastest QQ activity. AHL degradation proved to be enzymatic in most isolates (except for Stentrophomonas spp. and Pseudomonas sp.) as QQ activity could be destroyed by heat and/or proteinase K treatments. All QQ activity proved to be cell-bound except for Pseudomonas sp., where it could be detected in the supernatant. The results of aiiA gene homology analysis revealed the presence of aiiA gene encoding AHL lactonase in all examined isolates except Pseudomonas syringae and Enterobacter cloacae. The HXHXDH motif conserved in all AHL lactonases and considered to be essential for AHL degradation was detected in all AiiAs after sequence alignment. CONCLUSIONS: Some known and novel QQ bacteria were isolated from trouts and characterized in terms of enzymatic or nonenzymatic AHL degradation activity and their extracellular or intracellular location. In addition, an aiiA gene and its HXHXDH motif were detected in most isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: We could isolate and identify some novel QQ bacteria including Enterobacter hormaechei, Acinetobacter radioresistens and Citrobacter gillenii. The aiiA gene was detected for the first time in these strains as well as in Stenotrophomonas maltophilia. Our QQ isolates could be used for biocontrol of bacterial infections in aquaculture.

Morphological and molecular analyses of the spiruroid nematode, Falcaustra araxiana Massino, 1924 (= Spironoura araxiana) from the European pond turtle (Emys orbicularis).

There is little information on the phylogenetic position and life cycle of family Kathlaniidae. Falcaustra araxiana is a member of this family which infects the large intestine of the European pond turtle (Emys orbicularis). In the present study, morphological data and molecular analyses based on the 18S rDNA were performed on different types of F. araxiana originating from the large intestine and gastric nodules in the turtle. Morphological data revealed both larvae and adult stages in the gastric nodules. In addition, all nematodes recovered in the large intestine were adult worms. GenBank accession numbers KM200715 and KM200716 were provided for adult F. araxiana located in the intestine and stomach, respectively, of E. orbicularis. The results of sequencing proved that these two types are completely similar. Accordingly, it can be hypothesized that nodule formation is a part of the life cycle of the parasite or a survival strategy. Furthermore, F. araxiana develops to the adult stage in the gastric mucosa prior to migrating to the large intestine. Phylogenetic analysis revealed that F. araxiana unexpectedly branched away from other members of the superfamily Seuratoidea (Truttaedacnitis truttae, Cucullanus robustus and C. baylisi) and showed a closer relationship with Paraquimperia africana, a member of the Ascaridoidea. It seems that phylogenetic reconstruction for the present parasite needs more detailed morphology, life cycle and molecular studies.

A rare case of cerebral hydatidosis caused by a G1 genotype of Echinococcus granulosus in a cow from Iran.

Hydatidosis is a medically and veterinary important parasitic disease that is endemic in many parts of the world. Unilocular hydatid cysts may develop in almost any part of the body. Up to 70% of hydatid cysts are located in the liver, followed by 25% in the lungs. Cerebral hydatidosis is an uncommon manifestation of the disease, occurring in less than 1/1000 infected hosts, yet diagnosis does pose a problem. We have reported an exceptionally rare case of cerebral hydatidosis in cattle. This is the first report to describe the characteristic pathological features of the cerebral hydatidosis in cattle caused by the G1 genotype of Echinococcus granulosus. Genotypic analysis was performed on a hydatid cyst from a cow originating from southern Iran, based on the sequence analysis of the cox1 mitochondrial gene.

Characterization of Dicrocoelium dendriticum haplotypes from sheep and cattle in Iran based on the internal transcribed spacer 2 (ITS-2) and NADH dehydrogenase gene (nad1).

The present study assessed whether the genetic variation among different hosts (sheep and cattle) and geographical isolates (n= 28) of Dicrocoelium dendriticum from Iran is present based on mitochondrial (nad1) and ribosomal (ITS-2) DNA markers. Molecular analysis revealed the presence of at least ten and two distinct haplotypes in the NADH dehydrogenase gene (nad1) and internal transcribed spacer 2 (ITS-2), respectively. The nad1 and ITS-2 sequence data were deposited in GenBank under accession numbers, JX050110-134 and JQ966972-3. According to the results of our study, ND-D and ITS-A are established as being the predominant haplotypes of D. dendriticum in Iran. The Iranian isolates showed a higher intraspecific genetic diversity of 0-0.97% for nad1, compared to 0-0.42% for ITS-2. The alignment and comparison of nad1 and ITS-2 sequences revealed eight and one polymorphic sites, respectively. In the nad1 sequences, six were silent and two nucleotide substitutions were responsible for amino acid alterations. A phylogenetic analysis of the sequence data revealed that host associations and geographic location are not likely useful markers for D. dendriticum haplotype classification. Consequently, sequencing results obtained from the nad1 gene as a mitochondrial marker for the first time in this study would provide a valuable tool to analyse further molecular details of D. dendriticum worldwide.

Genetic characterization and phylogenetic analysis of Eimeria arloingi in Iranian native kids.

Among the 16 species of Eimeria from goats, Eimeria arloingi and Eimeria ninakohlyakimovae are regarded as the most pathogenic species in the world and cause clinical caprine coccidiosis. E. arloingi is known to be an important cause of coccidiosis in Iranian kids. Molecular analyses of two portions of nuclear ribosomal DNA (internal transcribed spacer1 (ITS1) and 18S rDNA) were used for the genetic characterization of the E. arloingi. Comparison of the sequencing data of E. arloingi obtained in the present study (ITS1: KC507793 and 18S rDNA: KC507792) with other Eimeria species in the GenBank database revealed a particularly close relationship between E. arloingi and Eimeria spp. from the cattle and sheep. The phylogram based on the ITS1 sequences shows that the E. arloingi, Eimeria bovis, and Eimeria zuernii formed a distinct group separate from the other remaining Eimeria spp. in cattle and poultry. In pairwise alignment, 18S rDNA sequence derived from E. arloingi showed 99% similarity to Eimeria ahsata with differences observed at only three nucleotides. This study showed that the ITS1 and 18S rDNA gene are useful genetic markers for the specific identification and differentiation of Eimeria spp. in ruminants.

The effect of polymorphism in gene of insulin-like growth factor-I on the serum periparturient concentration in Holstein dairy cows.

OBJECTIVE: To investigate the relationship between polymorphism within the 5'-untranslated region (5'-UTR) of IGF-I gene and its periparturient concentration in Iranian Holstein dairy cows. METHODS: Blood samples (5 mL, n = 37) were collected by caudal venipuncture from each animal into sample tubes containing the EDTA and DNA was extracted from blood. In order to measure IGF-I concentration the collection of blood samples (n = 111) was also done at 14 d before calving (prepartum), 25 and 45 d postpartum. RESULTS: We found evidence for a significant effect of C to T mutation in position 512 of IGF-I gene on its serum concentration in dairy cows in Iran. Cows with CC genotype had significantly higher concentration (Mean±SD) of IGF-I at 14 d prepartum (91.8±18.1) µg/L compared to those with TT genotype (73.3±14.4) µg/L (P=0.04). A significant trend (quadratic) was found for IGF-I concentration, as higher in CC cows compared to ones with TT genotype, during the 14 d before calving to 45 d postpartum (P=0.01). CONCLUSIONS: We concluded that C/T transition in the promoter region of IGF-I gene can influence the serum concentration of IGF-I in periparturient dairy cows.