Detection of bacteria in bovine ovarian follicular fluid.

The aim of this study was to examine the sterility of follicular fluid in large ovarian follicles in dairy cows. In all, 17 samples of paired follicular fluids and uterine contents collected from post-slaughtered dairy cows were cultured to detect aerobic and facultative anaerobic bacteria. Furthermore, the origin of the bacterial isolates from samples of follicular fluid and the uteri was also investigated using PFGE analysis. Follicular fluid concentrations of lipopolysaccharides were also determined. Of 17 uterine samples, 15 (88%) were detected as contaminated. In total, nine different bacterial genera and species were identified in the uterine and follicular fluid samples. Escherichia coli was the most prevalent bacterial species isolated from the uterine samples. Out of seven isolates of Staphylococcus aureus from the uterine samples, 6 (85%) were coagulase positive. Six isolates of Staphylococcus spp. were identified in 6 out of 17 follicular fluid samples (35%). Two out of six isolates were identified as Staphylococcus aureus (33%). Our results show that ovarian follicular fluid is not sterile in the bovine. The presence of Staphylococcus aureus in follicular fluid may partly explain the occurrence of infertility in some dairy cows. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present study show that ovarian follicular fluid is not sterile in bovines. The presence of Staphylococcus aureus in follicular fluid may partly explain the occurrence of infertility in some dairy cows.

DNA fingerprinting approaches to trace Escherichia coli sharing between dogs and owners.

AIMS: To determine the prevalence of cross-species sharing of Escherichia coli between healthy dogs and humans living in the same household. METHODS AND RESULTS: Two faecal E. coli isolates from 25 healthy dog-owner pairs and 16 healthy control humans were tested using three fingerprinting methods. The prevalence of within-household sharing of E. coli was 4, 8 and 8% using pulsed-field gel electrophoresis, randomly amplified polymorphic DNA and enterobacterial repetitive intergenic consensus-PCR analyses respectively. Within-household bacterial sharing was more prevalent than across-household sharing (P < 0·05). According to questionnaire analyses avoiding the dog-owner behaviours such as allowing a dog to kiss or lick the owner's face, sharing people food with dog and feeding it raw meat may decrease the chance of cross-species E. coli sharing. CONCLUSIONS: Direct contact between humans and dogs and environmental reservoirs may be important routes for cross-species sharing of bacteria. Good personal hygiene and appropriate veterinary care for pets can minimize this risk. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the importance of canine pathogenic E. coli reservoir hypothesis, close contacts between humans and dogs raises public health concerns. Determining the rate of cross-species bacterial sharing and confirm its accuracy by different fingerprinting techniques will help to find ways for reducing the economic impact of E. coli infections. This study support claims that public health concerns regarding the cross-species sharing of E. coli are warranted but this risk is minimal.

Distribution of virulence genes and multiple drug-resistant patterns amongst different phylogenetic groups of uropathogenic Escherichia coli isolated from patients with urinary tract infection.

A total of 85 Uropathogenic Escherichia coli (UPEC) isolates were screened against ceftiofur, oxacillin, nitrofurantoin and lincospectin using Kirby-Bauer disc diffusion method, following CLSI guidelines. Prevalence of virulent factor genes amongst the isolates was determined by PCR, using gene-specific primers against the different virulent factors. Statistical analysis of the data was performed using SPSS software. The prevalence of traT, ompT, Iss, malX and ibeA genes was 47.1%, 38.8%, 20%, 16.5% and 9.4%, respectively. The most prevalent gene in group A and D was traT, whilst in group B2 was Iss. The highest resistance has been shown against oxacillin (98.8%), followed by ceftiofur (77.6%), whilst resistance to lincospectin (2.4%) and nitrofurantoin (12.9%) had the lowest frequencies. Multidrug resistance was shown in 82.35% of the isolates, whilst this study recommend lincospectin and nitrofurantoin as choice drugs for treatment, but more investigation of the bacterial pathogenicity associated with urinary tract infection (UTI) may contribute to a better medical intervention.

Investigation of antibiotic resistance, virulence genes, and biofilm formation of Escherichia coli isolated from sheep feces in Shiraz industrial slaughterhouse, South of Iran.

Background: With the increase in human population, the consumption of livestock products such as sheep meat has also increased. Sheep are the reservoir and shedder of Escherichia coli that can be transmitted to humans. Aims: Characterization of fecal E. coli isolated from sheep in slaughterhouse. Methods: Stool specimens were collected from 30 apparently healthy sheep from different flocks in Shiraz industrial slaughterhouse. The resistance of E. coli isolates against 10 antibiotics was determined by disk diffusion method. The presence of three major extended spectrum beta-lactamase (ESBL) genes and five tetracycline resistance genes as well as seven virulence genes were investigated by polymerase chain reaction (PCR) technique. Using the microtiter plate method, the biofilm formation ability of E. coli isolates was investigated. Results: The highest frequency of resistance was to amoxicillin (100%) followed by tetracycline (25%). All E. coli isolates were susceptible to gentamicin and nitrofurantoin, and only one isolate was resistant to the tested third-generation cephalosporins. Multidrug resistance phenotype was observed in 16.7% of the isolates. bla TEM (25%) was the most prevalent ESBL gene and tetA (62.5%) was the most prevalent tetracycline resistance gene in the isolates. crl, csgA, fimH, and bcsA genes were present in all isolates, and the prevalence of papC and afa genes was 95.8% and 83.3%, respectively. In total, 62.5% of the isolates were biofilm producers. Conclusion: According to the concept of One Health, the presence of virulent antibiotic-resistant biofilm producing strains of E. coli in sheep is a risk to public health.

Identification of Mycoplasma capricolum subspecies capripneumoniae and Mycoplasma arginini by culture, PCR, and histopathology in pneumonic lungs of slaughtered goats in Mashhad, Iran.

Background: A number of Mycoplasma spp., often referred to as the Mycoplasma mycoides (Mm) cluster can produce respiratory tract infections in goats; however, only Mycoplasma capricolum subspecies capripneumoniae (Mccp) is considered to causecontagious caprine pleuropneumonia. Aims: Isolation and identification of M. capricoluum subspecies capripneumoniae and M. arginini from the pneumonic lungs of slaughtered goats and their association with pathological changes. Methods: Lungs of 2000 goats slaughtered at an industrial abattoir in Mashhad, Iran, were examined for the presence of gross pneumonic lesions. Fifty affected lungs were selected for pathology, culture, and molecular (PCR) studies for the presence of Mycoplasma species. DNA was extracted from lung tissue samples and replicated using genus and species specific primers for Mycoplasma. Results: Grossly, consolidation and dark red to grey discoloration in the cranioventral to caudal lobes in fibrinopurulent bronchopneumonia and rubbery texture associated with rib impressions on the costal surfaces of the diaphragmatic lobes in interstitial pneumonia were observed. Histopathologically, bronchointerstitial pneumonia in 40 (80%), and fibrinopurulent bronchopneumonia in 10 (20%) of affected goats were diagnosed. The evidence of Mycoplasma growth such as turbidity and Mycoplasma colonies on the Mycoplasma agar plates was observed in 2 (4%) of samples. Genus-specific Mycoplasma DNA was identified in 11 (22%) of samples. Of them, 3 (6%) and 3 (6%) of tissue lung samples were positive for M. capricolum subspecies capripneumoniae and M. arginini, respectively, by PCR. Conclusion: Our results showed that M. capricolum subspecies capripneumoniae and M. arginini were the two agents that can involve lung consolidation and pneumonia in goats.

Biofilm formation by uropathogenic Escherichia coli: a complicating factor for treatment and recurrence of urinary tract infections.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) are the principal cause of urinary tract infections (UTIs) which can be either hospital- or community-acquired. The most crucial factor in the persistence and recurrence of UTIs is the biofilm formation ability of UPEC, which protects them against antimicrobial treatment. AIM: To investigate the genetic relatedness, biofilm formation ability, and biofilm-related genes in UPEC isolated from hospital- and community-acquired UTI patients. METHODS: In vitro biofilm formation ability of 100 UPEC isolates, collected from the urine samples of 49 inpatients and 51 outpatients with UTIs, was assessed by the microtitre plate method. The association between the presence of fimH, papC, sfa/focDE, csgA, crl, afa, flu, and bcsA genes and biofilm formation ability of UPEC was statistically analysed. The genetic relatedness of UPEC isolates was evaluated by enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). FINDINGS: Overall, 99% of the UPEC isolates showed in vitro biofilm formation ability, and 27% of the isolates were moderate to strong biofilm producers. Only the presence of sfa/focDE gene was significantly associated with moderate and strong biofilm formation by the UPEC isolates. Analysis of dendrograms revealed higher genetic similarities among UPEC isolates of inpatients compared with outpatients. CONCLUSION: Based on the results, selection of effective therapeutic approaches, which can affect both biofilm formation and enclosed UPEC, is important for preventing recurrent UTIs. The common UPEC clones among inpatients in different hospital units emphasize the need for more rigid control measures to prevent the spread of UPEC in hospitalized patients and to reduce the occurrence of hospital-acquired UTIs.

Identification of Mycoplasma ovipneumoniae and Mycoplasma arginini in sheep with pneumonia in North East of Iran.

BACKGROUND: Pneumonia due to Mycoplasma infections can cause serious health problems and economic losses in small ruminants industry. AIMS: The aim of this study was isolation and identification of Mycoplasmas in sheep naturally infected with pneumonia in Northeastern Iran. METHODS: This study used histopathology, culture and polymerase chain reaction (PCR) to examine samples from 50 lungs of sheep naturally infected with Mycoplasmas. RESULTS: Grossly, irregular consolidation with lobular or lobar to diffuse pattern in the cranioventral to caudal lobes of affected lungs were observed. Histopathologically, bronchointerstitial pneumonia in 38 (76%), and purulent to fibrinopurulent bronchopneumonia in 12 (24%) affected sheep were diagnosed. DNA was extracted from lung tissue samples and replicated using genus and species specific primers for Mycoplasma. Mycoplasma growth was observed in 3 (6%) of a total of 50 lung samples. Genus-specific Mycoplasma DNA was identified by PCR in 12 (24%) of samples. Two (4%) and 7 (14%) samples of these 12 cases were positive for reaction with species-specific primers of Mycoplasma ovipneumoniae and Mycoplasma arginini, respectively. CONCLUSION: Our results showed that M. ovipneumoniae and M. arginini were the two agents that can be involved in inducing lung consolidation and pneumonia in sheep and PCR was more successful than the culture in detecting Mycoplasmas.

Identification, cloning and sequencing of Escherichia coli strain chi1378 (O78:K80) iss gene isolated from poultry colibacillosis in Iran.

AIMS: To identify, clone and sequence the iss (increased serum survival) gene from E. coli strain chi1378 isolated from Iranian poultry and to predict its protein product, Iss. METHODS AND RESULTS: The iss gene from E. coli strain chi1378 was amplified and cloned into the pTZ57R/T vector and sequenced. From the DNA sequence, the Iss predictive protein was evaluated using bioinformatics. Iss from strain chi1378 had 100% identity with other E. coli serotypes and isolates from different origins and also 98% identity with E. coli O157:H7 Iss protein. Phylogenetic analysis showed no significant different phylogenic groups among E. coli strains. CONCLUSIONS: The strong association of predicted Iss protein among different E. coli strains suggests that it could be a good antigen to control and detect avian pathogenic E. coli (APEC). SIGNIFICANCE AND IMPACT OF THE STUDY: Because the exact pathogenesis and the role of virulence factors are unknown, the Iss protein could be used as a target for vaccination in the future, but further research is required.

Co-relation of estrous cycle phases with uterine bacterial and fungal flora in non-pregnant female laboratory rabbits.

This study was designed to investigate the relationship between the estrous cycle phases with uterine bacterial and fungal flora in non-pregnant female rabbits. Thirty laboratory mature multiparous rabbits were used for this purpose. Samples from uterine lavage for culture of bacteria and fungi were collected at different stages of estrous cycle (based on vaginal cytology), and histopathological observations were evaluated based on the scoring system used for defining the infection of the uterus. Various types of bacteria and fungi were isolated from rabbits at all stages of estrous cycle. The widest variety of bacteria and fungi was isolated at Di-estrous stage and the lowest variety was detected at estrous stage. Klebsiella oxytoca as well as yeast have been isolated at all stages of estrous cycle. This study showed that infection with K. oxytoca and yeast had no relationship with different stages of estrous cycle but other bacteria and fungus were associated with one or more stages of the estrous cycle in rabbits.

Brucella melitensis and Mycobacterium tuberculosis depict overlapping gene expression patterns induced in infected THP-1 macrophages.

Pathogens infecting mammalian cells have developed various strategies to suppress and evade their hosts' defensive mechanisms. In this line, the intracellular bacteria that are able to survive and propagate within their host cells must have developed strategies to avert their host's killing attitude. Studying the interface of host-pathogen confrontation can provide valuable information for defining therapeutic approaches. Brucellosis, caused by the Brucella strains, is a zoonotic bacterial disease that affects thousands of humans and animals around the world inflicting discomfort and huge economic losses. Similar to many other intracellular dwelling bacteria, infections caused by Brucella are difficult to treat, and hence any attempt at identifying new and common therapeutic targets would prove beneficial for the purpose of curing infections caused by the intracellular bacteria. In THP-1 macrophage infected with Brucella melitensis we studied the expression levels of four host's genes, i.e. EMP2, ST8SIA4, HCP5 and FRMD5 known to be involved in pathogenesis of Mycobacterium tuberculosis. Our data showed that at this molecular level, except for FRMD5 that was downregulated, the other three genes were upregulated by B. melitensis. Brucella melitensis and M. tuberculosis go through similar intracellular processes and interestingly two of the investigated genes, i.e. EMP2 and ST4SIA8 were upregulated in THP-1 cell infected with B. melitensis similar to that reported for THP-1 cells infected with M. tuberculosis. At the host-pathogen interaction interface, this study depicts overlapping changes for different bacteria with common survival strategies; a fact that implies designing therapeutic approaches based on common targets may be possible.

Identification of Helicobacter spp. in gastrointestinal tract, pancreas and hepatobiliary system of stray cats.

The aim of the present study was to determine the presence of Helicobacter species in different parts of gastrointestinal tract, hepatobiliary system and pancreas of stray cats. Six different sites at the level of genus, gastric (H. heilmannii and H. felis) and enterohepatic species of Helicobacter were investigated in six cats using species-specific primers by polymerase chain reaction (PCR). Interestingly, DNA of enterohepatic spp. was detected in 1/6 duodenum, 2/6 colon and 1/6 pancreas specimens. Results of sequencing revealed that all of these four positive samples belong to Helicobacter canis. While cats have not been considered as a potential zoonotic danger for non-pylori Helicobacter infections, the results of current study show prompt re-evaluation of that view. To the best of our knowledge, this is the first study about distribution of Helicobcater spp. in gastrointestinal tract of cats.

The relationship between growth hormone polymorphism and growth hormone receptor genes with milk yield and reproductive performance in Holstein dairy cows.

The aim of this study was to investigate the potential association between growth hormone GH/AluI and growth hormone receptor GHR/AluI polymorphisms with milk yield and reproductive performances in Holstein dairy cows in Iran. Blood samples of 150 Holstein cows were collected and their genomic DNA was extracted using Gene-Fanavaran DNA extracting kit. Fragments of the 428 bp of exon 5 growth hormone (GH) gene and the 342 bp of exon 10 growth hormone receptor (GHR) gene were amplified using the polymerase chain reaction (PCR) method. PCR products were digested by the AluI restriction enzyme and electrophoresed on 3% agarose gel. Continuous and categorical data were analyzed using linear mixed models through Proc MIXED and logistic regression models through Proc GENMOD of SAS software, respectively. The results showed no relationship between the examined traits and GH/AluI or GHR/AluI genes. A significant relationship was found between GH/AluI polymorphism and dystocia, but the presence of the GH-L allele reduced the incidence of dystocia. The results suggest that the GH-LL genotype reduces dystocia probably by affecting the release of growth hormone; nevertheless, further studies will be needed to examine the relationship between dystocia and GH genotypes.

Association between the enterotoxin production and presence of Coa, Nuc genes among Staphylococcus aureus isolated from various sources, in Shiraz.

The present study was aimed to identify the frequency of coagulase (Coa) and thermonuclease (Nuc) genes and Staphylococcal enterotoxin A (Sea) production among Staphylococcus aureus isolated from various sources in Shiraz. Moreover, the correlation between the Sea gene and coagulase and thermonuclease enzymes is also considered. A total of 100 S. aureus were isolated from various sources including 40 humans, 30 animals and 30 food samples by the routine biochemical tests. The frequency of Coa, Nuc and Sea genes was evaluated by PCR assay. Correlation among those genes was finally evaluated by statistical analysis. The PCR results showed that the prevalence of Coa, Nuc and Sea genes was 91%, 100% and 14%, respectively. The evaluation of the enterotoxin production indicated that 78.6% of the Sea gene was expressed. The presence of enterotoxin A was not necessarily correlated to the production of toxin. As a final conclusion to detect the enterotoxigenic strains, both genotypic and phenotypic methods are highly recommended.