Molekularni dokaz infekcija uzrokovanih hemoplazmama u pasa u južnom Iranu i njihovo razlikovanje na osnovi polimorfizma dužine restrikcijskog fragmenta.

Two hemoplasma species are known in dogs: Mycoplasma haemocanis (Mhc) and Candidatus Mycoplasma haematoparvum (CMhp). The aim of the present study was to develop a novel restriction fragment length polymorphism (RFLP)-PCR method based on the 16S rDNA gene, using endonuclease Hind III, for detection and differentiation of canine hemoplasmas. Also, analysis of risk factors, clinical features and hematologic changes of positive cases was performed in dogs living in the Shiraz area of Iran. Blood samples were collected from anemic (packed cell volume (PCV) ≤35; n = 26) and control dogs (PCV >35; n = 27) and were examined for the presence of canine hemoplasmas, using RFLP-PCR and 16S rDNA Sanger sequencing. The presence of Mhc (4 out of 53 cases; 7.5%) and CMhp (3 out of 53 cases; 5.7%) was confirmed by RFLP analysis of the amplified 16S rDNA and sequencing. No association was found between hemoplasma infection and anemia, health status, age, breed, gender, type of housing or the presence of other dogs in this study. Only the platelet number in Mhc infected dogs was statistically higher compared to CMhp positive and hemoplasma negative dogs. The present report documents the occurrence of Mhc and CMhp in southern Iran, and these hemotropic Mycoplasma infections must be expected even in the absence of clinical symptoms or hematologic abnormalities in dogs. For the first time, it has been indicated that RFLP-PCR assay is able to successfully distinguish hemotropic Mycoplasma in dogs.U pasa su poznate dvije vrste hemoplazama: Mycoplasma haemocanis (Mhc) i Candidatus Mycoplasma haematoparvum (CMhp). Cilj je ovog istraživanja bio razviti novu metodu za dokaz i razlikovanje pasjih hemoplazama temeljenu na određivanju polimorfizma dužine restrikcijskog fragmenta (PDRF) gena 16S rDNA uporabom endonukleaze Hind III. Analizirani su i rizični čimbenici, kliničke osobitosti i hematološke promjene u inficiranih pasa na području Shiraza u Iranu. Uzorci krvi bili su prikupljeni od anemičnih (hematokrit ≤35; n = 26) i kontrolnih pasa (hematokrit >35; n = 27) te pretraženi na prisutnost pasjih hemoplazama metodom RFLPPCR i Sangerovom metodom sekvenciranja 16S rDNA. Prisutnost Mhc (4 od 53 slučaja; 7,5%) i CMhp (3 od 53 slučaja; 5,7%) bila je potvrđena analizom polimorfizma restrikcijskog fragmenta 16S rDNA i sekvenciranjem. Nije ustanovljena veza između infekcije hemoplazmama i anemije te zdravstvenog stanja, dobi, pasmine, spola, načina držanja i prisutnosti drugih pasa. Jedino je broj trombocita u pasa inficiranih vrstom Mycoplasma haemocanis bio statistički značajno veći od onih inficiranih Candidatus Mycoplasma haematoparvum i pasa negativnih na hemoplazme. Ovo izvješće potkrepljuje prisutnost Mhc i CMhp u južnom Iranu. Infekcije hemotropnim mikoplazmama mogu se očekivati i u pasa bez kliničkih znakova ili hematoloških poremećaja. Prvi put je pokazano da se metodom RFLP-PCR mogu uspješno razlikovati hemotropne mikoplazme u pasa

Istraživanje gena za otpornost na antibiotik tetraciklin u izolata bakterije Escherichia coli izdvojenih iz tovnih pilića u Iranu.

The tetracycline resistance of E. coli isolates (n = 300) from broiler chickens was investigated in three stages of the rearing period (one-day-old chicks, thirty-day-old chickens and one day before slaughter). Tetracycline resistance genes (tet(A), tet(M), tet(O) and tet(S)) were investigated among 120 tetracycline resistant E. coli isolates. Tetracycline resistance at the three stages of sampling was 67, 90 and 94%, respectively. Of the 120 tetracycline resistant E. coli isolates, 68 (59%) carried the tet(A) resistance gene. The tet(A) resistance gene was present in 32.5% (13/40) of E. coli isolated from one-day-old chicks, 65% (26/40) of E. coli isolated from thirty-day-old chickens and 72.5% (29/40) of E. coli isolated from the chickens on the day before slaughter. None of the tested isolates contained tet(M), tet(O) or tet (S). Tetracycline resistance was relatively high in E. coli isolated from one-day-old chicks, suggesting a high prevalence of tetracycline resistance in the preliminary gut flora of these broiler chicks. Significant increases in the resistance rate of E. coli isolates were found during the 2nd and 3rd rearing period. Tet(A) was the only detected tet resistance gene in these E. coli isolates. The rise of the tet(A) resistance gene during the rearing of broilers is alarming because this plasmid mediated tet gene can be transmitted to other pathogenic and commensal bacteria in the poultry industry.Istražena je otpornost na tetraciklin izolata vrste E. coli (n = 300) izdvojenih iz tovnih pilića triju dobnih skupina (jednodnevnih, 30 dnevnih i pilića jedan dan prije klanja). Ukupno je 120 izolata E. coli otpornih na tetraciklin bilo pretraženo na prisutnost gena za otpornost na tetraciklin (tet(A), tet(M), tet(O) i tet(S)). Otpornost na tetraciklin u izolatima iz jednodnevnih pilića iznosila je 67%, u izolatima iz 30 dnevnih pilića 90%, a u onih izdvojenih iz pilića dan prije klanja 94%. Od 120 izolata otpornih na tetraciklin, 68 (59%) nosilo je gen tet(A). Gen tet(A) dokazan je u 32,5% (13/40) izolata E. coli iz jednodnevnih pilića, u 65% (26/40) iz 30-dnevnih pilića i 72,5% (29/40) iz pilića dan prije klanja. Nijedan od pretraženih izolata nije sadržavao tet(M), tet (O) or tet(S). Otpornost na tetraciklin bila je relativno visoka u izolata iz jednodnevnih pilića, što upućuje na zaključak o visokoj prevalenciji otpornosti primarne crijevne flore na tetraciklin. Značajno povećanje stope otpornosti izolata E. coli ustanovljeno je tijekom 2. i 3. uzgojnog razdoblja. Tet(A) bio je jedini dokazan tet gen u pretraženih izolata. Povećana učestalost gena otpornosti tet(A) tijekom uzgojnog razdoblja tovnih pilića je zabrinjavajuća jer se plazmid prenositelj gena tet može prenijeti na druge patogene i komenzalne bakterije u peradi

Molekularni dokaz infekcija uzrokovanih hemoplazmama u pasa u južnom Iranu i njihovo razlikovanje na osnovi polimorfizma dužine restrikcijskog fragmenta.

Two hemoplasma species are known in dogs: Mycoplasma haemocanis (Mhc) and Candidatus Mycoplasma haematoparvum (CMhp). The aim of the present study was to develop a novel restriction fragment length polymorphism (RFLP)-PCR method based on the 16S rDNA gene, using endonuclease Hind III, for detection and differentiation of canine hemoplasmas. Also, analysis of risk factors, clinical features and hematologic changes of positive cases was performed in dogs living in the Shiraz area of Iran. Blood samples were collected from anemic (packed cell volume (PCV) ≤35; n = 26) and control dogs (PCV >35; n = 27) and were examined for the presence of canine hemoplasmas, using RFLP-PCR and 16S rDNA Sanger sequencing. The presence of Mhc (4 out of 53 cases; 7.5%) and CMhp (3 out of 53 cases; 5.7%) was confirmed by RFLP analysis of the amplified 16S rDNA and sequencing. No association was found between hemoplasma infection and anemia, health status, age, breed, gender, type of housing or the presence of other dogs in this study. Only the platelet number in Mhc infected dogs was statistically higher compared to CMhp positive and hemoplasma negative dogs. The present report documents the occurrence of Mhc and CMhp in southern Iran, and these hemotropic Mycoplasma infections must be expected even in the absence of clinical symptoms or hematologic abnormalities in dogs. For the first time, it has been indicated that RFLP-PCR assay is able to successfully distinguish hemotropic Mycoplasma in dogs.U pasa su poznate dvije vrste hemoplazama: Mycoplasma haemocanis (Mhc) i Candidatus Mycoplasma haematoparvum (CMhp). Cilj je ovog istraživanja bio razviti novu metodu za dokaz i razlikovanje pasjih hemoplazama temeljenu na određivanju polimorfizma dužine restrikcijskog fragmenta (PDRF) gena 16S rDNA uporabom endonukleaze Hind III. Analizirani su i rizični čimbenici, kliničke osobitosti i hematološke promjene u inficiranih pasa na području Shiraza u Iranu. Uzorci krvi bili su prikupljeni od anemičnih (hematokrit ≤35; n = 26) i kontrolnih pasa (hematokrit >35; n = 27) te pretraženi na prisutnost pasjih hemoplazama metodom RFLPPCR i Sangerovom metodom sekvenciranja 16S rDNA. Prisutnost Mhc (4 od 53 slučaja; 7,5%) i CMhp (3 od 53 slučaja; 5,7%) bila je potvrđena analizom polimorfizma restrikcijskog fragmenta 16S rDNA i sekvenciranjem. Nije ustanovljena veza između infekcije hemoplazmama i anemije te zdravstvenog stanja, dobi, pasmine, spola, načina držanja i prisutnosti drugih pasa. Jedino je broj trombocita u pasa inficiranih vrstom Mycoplasma haemocanis bio statistički značajno veći od onih inficiranih Candidatus Mycoplasma haematoparvum i pasa negativnih na hemoplazme. Ovo izvješće potkrepljuje prisutnost Mhc i CMhp u južnom Iranu. Infekcije hemotropnim mikoplazmama mogu se očekivati i u pasa bez kliničkih znakova ili hematoloških poremećaja. Prvi put je pokazano da se metodom RFLP-PCR mogu uspješno razlikovati hemotropne mikoplazme u pasa

Histopathological and Molecular Evaluation of the Experimen-tally Infected Goats by the Larval Forms of Taenia multiceps

Background: Introduction of Taenia multiceps and T. gaigeri as two separate species have been recognized mainly on morphological grounds. This experimental study was conducted to test whether cerebral and non-cerebral forms of Coenurus cerebralis belong to one origin or they are originated from two different tape worms. Methods:  Two groups of dogs were infected with the cerebral and muscular sources of the coenuri cysts. About two months later the eggs were collected from the fecal samples to be used to experimentally infect other healthy goats. Histopathological and molecular evaluation was conducted in two groups of goats that were challenged with T. multiceps eggs obtained from the infected dogs by brain and muscular sources of coenuri cysts in School of Veterinary Medicine of Shiraz University, Shiraz, Iran in 2015. All aberrant sites of predilection of the metacestode in goats were muscles, heart, diaphragm and lungs. The brain and spinal cord were carefully dissected and examined but the cysts were not found in these locations. In addition, the molecular genetic markers of mitochondrial DNA (CO1 and ND1) were applied to resolve the questionable relationship between T. multiceps and T. gaigeri. Results: The larval stages of T. multiceps in brain and in other aberrant sites, which showed similar morphological criteria, were monophyletic species. Conclusion: Therefore, T. gaigeri must be considered taxonomically invalid

Antimicrobial Resistance and Incidence of Integrons in Aeromonas Species Isolated from Diseased Freshwater Animals and Water Samples in Iran

Aeromonas spp. is one of the major pathogens of freshwater animals. There has been little research on the genetics of antimicrobial resistance associated with it in Iranian aquaculture. To remedy this lack in research, 74 multi-drug-resistant Aeromonas spp. were isolated from farmed diseased carp, trout, sturgeon, ornamental fish, crayfish, and corresponding water samples and examined for genomic integron sequences. Class 1 integrons, containing seven types of integron cassette arrays (dfrA1-aadA1, dfrA1-orfC, dfrA12-aadA2, dfrA12-orfF-aadA2, dfrA15, dfrB4-catB3-aadA1, aac(6’)-Ib-cr-arr3-dfrA27) were found in 15% of the resistant isolates; no class 2 integrons were detected in any of the resistant isolates. As some tested isolates were resistant to more than two groups of antibiotics, our results demonstrated that freshwater animals in Iran could be a source of multiply drug-resistant Aeromonas spp. This finding suggests that the origin of the antimicrobial resistance of these animals be placed under increased surveillance in the future and that the use of antimicrobials be limited in aquaculture

Vaginal Fornix Discharge Cellularity and Its Leukocyte Esterase Activity for Diagnosis of Endometritis in Dairy Cows

The objective of the present study was to evaluate the application of some strip test markers (i.e., leukocyte esterase (LE) activity, protein, nitrate and pH) for diagnosis of endometritis in dairy cows using vaginal fornix discharge. Also, the total white blood cell count (t-WBC/l) of this secretion and degenerative changes of neutrophils in cervical cytology were used as alternative methods to predict progression of the endometritis severity. Holstein cows (n=215) between 30-40 days in milk (DIM) were included and examined. Giemsa-stained smear was prepared from cervical mucus. Cervical cytology test was considered as reference screening method for the detection of subclinical endometritis. The LE activity and t-WBC in the vaginal fornix discharge of subclinical endometritis cows were significantly higher than those from healthy cows. Sensitivity and specificity were 78% and 73% for LE10 activity (10 minutes after contacting with discharges) and 60% and 69% for t-WBC (cut off point=210 cells/l) for diagnosis of subclinical endometritis, respectively. There was a good agreement between LE10 activity, t-WBC and cervical cytology test with a Kappa coefficient of 0.4 and 0.42, respectively (P<0.0001). Total WBC count in discharge and degenerative neutrophils (DN) percentages increase simultaneously with the degree and severity of endometritis. There was a highly significant (P<0.01) correlation between t-WBC and some reagent strip test markers (LE activity, protein and nitrate) in clear discharge of studied cows. In conclusion, the present results suggest the LE activity and t-WBC in vaginal fornix discharge could be used as non-invasive reliable and valid methods for screening of subclinical endometritis in postpartum dairy herds

Genetic Characterization of Nematodirella cameli Based on 18S rDNA and Cytochrome c Oxidase Subunit 1 (CO1)

To determine the phylogenic position and genetic diversity of Nematodirella cameli two portions of nuclear ribosomal DNA, 18S rDNA and mitochondrial DNA gene, the subunit 1 of cytochrome C oxidase gene (CO1) were sequenced and compared with those previously reported for other nematodes in Trichostrongylina. The phylogenetic trees constructed based upon the 18S rDNA sequences, yielded strong support for close relationship between the N. cameli and Nematodirus battus, with a high bootstrap value of 100%. In the present research, the level of sequence polymorphism among N. cameli isolates was higher for CO1 with 32 polymorphic sites compared to 18S rDNA sequence. Accordingly, molecular assays based on CO1 mitochondrial marker, demonstrated the existence of at least 11 distinct haplotypes (accession nos. JX305966 to JX305976) with an intraspecific diversity of 3-7% in Iran. Whereas, all of N. cameli samples examined herein (n=11), had a unique 18S sequence (accession no. JX305977). In addition, N. cameli CO1 sequences found in this study showed maximum identities to Haemonchus (88%) and Ostertagia (87%) in BLAST analysis for existing Trichostrongylina sequences. Further information is necessary to infer interspecific and intraspecific phylogenetic relationships between genera and species in Trichostrongylina. This study describes for the first time the nuclear 18S rDNA and mitochondrial CO1 sequence data from Nematodirella cameli species

Molecular Differentiation of Fasciola Species and Characterization of Genetic Diversity of F. gigantica Using NADH Dehydrogenase I (ND1) Gene in the Endemic Areas of Iran

  Background: Fasciola hepatica and F. gigantica are the causative agents of fasciolosis in domestic animals and humans. Based on the morphometric criteria, differential diagnosis between them is problematic. In addition, intermediate forms of Fasciola have been found in Iran, which makes the differentiation more difficult. The aim of the present study was to provide molecular evidence for the existence of F. gigantica in Iran using sequencing analysis of ND1 and PCR-RFLP analysis of ITS2 regions and to study the intraspecies variations of F. gigantica based on mitochondrial ND1 gene polymorphism. Methods:Forty Fasciola spp. samples collected from four distinct provinces (Fars, Khuzestan, Gilan, Khorasan Razavi) in Iran were collected for morphological and molecular characterization. In molecular method, PCR-RFLP analysis of ITS2 us-ing pagI restriction enzyme was used as a screening approach for F. gigantica differ-entiation. Then mitochondrial DNA sequence variations in the ND1 gene were used for phylogenetic analysis. Results:Based on the morphometric criteria and RFLP analysis, 14 parasitic sam-ples were initially identified to be F. gigantica. Phylogenetic results showed that there are at least 10 different genotypes of F. gigantica in Iran, which are different from those existing in the GenBank. Twenty-six points out of 410 base pairs of se-quenced ND1 gene in 10 varieties of F. gigantica were diagnosed to be polymorphic. From 26 points of polymorphism, only eight resulted in the post-translational ami-no acid changes in ND1 gene product structure. Conclusion:Data revealed noticeable genetic diversity (up to 4.63%) between different varieties of F. gigantica in Iran

Characterization of single nucleotide polymorphism in the 5'-untranslated region (5'-UTR) of Lactoferrin gene and its association with reproductive parameters and uterine infection in dairy cattle

Uterine infection is one of the reproductive diseases that can have disturbing postpartumuterine health in cattle. Therefore, identification of resistant genotypes to uterine infection isimportant. Lactoferrin (LF) is one of the major antimicrobial compounds in the normal uterinedischarges of cows. We hypothesized that allelic diversity in LF gene may contribute tosusceptibility or resistance to uterine infection. We investigated the single nucleotidepolymorphism genes identified in the 5' untranslated region (5'-UTR, position +32) of the LF geneusing Allele-specific PCR method in cows with and without uterine infection. Blood samples werecollected from 89 multiparous Holstein dairy cows with a history of uterine infection (n = 51), andcows without disease as the control group (n= 38). The results indicated the presence of differentproportion of polymorphisms (G > C) in the 5'-UTR area of cows in the all groups. The results ofAllele specific PCR was in complete agreement with sequencing method. Statistical analysis did notshow any statistically significant correlation between disease and SNP in 5'-UTR. While, there was asignificant difference in the mean of reproductive parameters of cows without polymorphismcompare to those of with SNP in 5'-UTR. Cows with +32:CC genotype and +32:GC genotype (cowswith SNP in UTR) had lower average of services per conception and days open compared to cowswith the +32:GG genotypes. However, no significant difference in the calving to first service wasfound between these genotypes. Further studies will be required to determine critical SNPs in LFgene and status of the risk of uterine infection and embryo survival in cows

Effect of catecholamine stress hormones (dopamine and norepinephrine) on growth, swimming motility, biofilm formation and virulence factors of Yersinia ruckeri in vitro and an in vivo evaluation in rainbow trout

In this study, we evaluated the impact of the catecholamines on growth, swimming motility, biofilm formation and some virulence factors activities of pathogenic Yersinia ruckeri. Norepinephrine and dopamine (at 100 mu M) significantly increased the growth of Y. ruckeri in culture media containing serum. An increase in swimming motility of the pathogen was found following the exposure to the hormones; however, no effect was seen on caseinase, phospholipase and haemolysin productions. Further, antagonists for the catecholamine receptors were observed to block some of the influences of the catecholamines. Indeed, the effects of catecholamines were inhibited by chlorpromazine (the dopaminergic receptor antagonist) for dopamine, labetalol (alpha-and beta-adrenergic receptor antagonist) and phenoxybenzamine (the alpha-adrenergic receptor antagonist) for norepinephrine, but propranolol (the beta-adrenergic receptor antagonist) showed no effect. Pretreatment of Y. ruckeri with the catecholamines resulted in a significant enhancement of its virulence towards rainbow trout and the antagonists could neutralize the effect of the stress hormones in vivo. In summary, our results show that the catecholamines increase the virulence of Y. ruckeri which is pathogenic to trout through increasing the motility, biofilm formation and growth