Polymerase Chain Reaction Examination Research Articles

Influence of land use and meteorological factors on the spatial distribution of Toxocara canis and Toxocara cati eggs in soil in urban areas

Soil which has been contaminated by Toxocara spp. eggs is considered as one of the main infection sources of Toxocariasis in animals and humans. The present study conducted a detailed investigation into the spatial patterns of Toxocara canis (T. canis) and Toxocara cati (T. cati) eggs in soil in urban area of northeastern Mainland China, and assessed the inter-relationships between meteorological factors, land use and the distribution of the Toxocara spp. eggs. Polymerase chain reaction (PCR) was used for the determination of T. canis and T. cati eggs contamination in soil samples. Between April 2014 and May 2015, 9420 soil samples were subjected to PCR examination and 7027 sheep (74.6%) were determined to be positive for T. canis and T. cati eggs. Subsequently, we evaluated the effect of land use, and meteorological factors on the spatial distribution of T. canis and T. cati eggs based on a maximum entropy model. Jackknife analysis revealed that the area of residential land, wood and grass land and precipitation may influence the occurrence of T. canis and T. cati eggs in soil. Our findings indicate that land use and meteorological factors may be important variables affecting transmission of Toxocariasis and should be taken into account in the development of future surveillance programmes for Toxocariasis.

4. Detection of Toxoplasma gondii infection by Polymerase Chain Reaction (PCR) and Histological Examination on Balb/c Mice

The purpose of this research was to compare the use of PCR method and histological examination to diagnose toxoplasmosis in tissues of Balb/c mice infected with sporulated oocysts through drinking water. A total of 20 male Balb/c mice aged approximately 2 months were used in this experiment. Each mouse was infected with 1x103 Toxoplasma gondii tachyzoites intraperitoneally. Tissue samples (liver, lung, heart, kidney, and brain) were collected from 5 mice on day 1, day 5, day 7, and day 9 after infection. Samples were then examined by PCR and histological methods. The data collected were analyzed descriptively. The results showed that PCR method was more sensitive than histological examination. PCR examination using primer invitrogen gen can amplify DNA T. gondii at 436 bp of the samples from liver, lung, heart and brain on Day 7 and Day 9 after infection. The histological examination showed that the cyst of toxoplasma was found in the brain while mononuclear cells infiltration was found in other internal organs.

COMPARATIVE STUDY ON THE INTENSITY OF Mycobacterium leprae EXPOSURE BETWEEN HOUSEHOLD AND NONHOUSEHOLD CONTACT OF LEPROSY

Leprosy stills a public health problem in West Sulawesi which has a Case Detection Rate (CDR) around 43.69/100.000 population. Household contacts of leprosy are a high risk group to be infected, due to droplet infection mode of transmission of the disease. A nose swab examination and serological study was conducted to detect exposure of M. leprae of people who live in leprosy endemic area. Detection of M. leprae in the nasal cavity will represent the exposure rate from outside and the measurement of specific antibody is represented the result of exposure to the immune system. Two group of inhabitants (30 household contacts of leprosy and 30 nonhousehold contacts) were involved in the study. They live in Banggae district, a leprosy endemic area of Majene Regency, West Sulawesi. Sixty nose swab samples and sixty capillary blood samples from the same invidividuals of the two groups were collected and sent to Leprosy laboratory of the Institute of Tropical Disease, Airlangga University Surabaya. A Polymerase Chain Reaction (PCR) was performed to the nose swab samples for detection of M. leprae. The blood samples were examined serologically to measure the level of anti PGL-1 antibody. PCR examination of nose swab samples showed 1/30 positive result in the household contact group and also 1/30 positive result in non-household contact of leprosy (statistically no significant difference, p > 0.05). Serological study showed higher sero-positive result in the household contact group (15/30 or 50%) compared to non-household contact (11/30 or 36%), but statistical calculation revealed no significant difference between the two groups (p > 0.05) on sero-positive results of leprosy. It is concluded that household and non-household contact in leprosy have the same risk to be affected by the disease. The term of household and non-household contact need to be redefined. The possible role of exposure from the environment was also discussed, especially from non-human resource of M. leprae.

Expression of macrophage migration inhibitory factor and CD74 in the inner ear and middle ear in lipopolysaccharide-induced otitis media

Conclusion: Significant expression of macrophage migration inhibitory factor and its receptor (CD74) was observed in both the middle ear and inner ear in experimental otitis media in mice. Modulation of macrophage migration inhibitory factor and its signaling pathway might be useful in the management of inner ear inflammation due to otitis media.Objectives: Inner ear dysfunction secondary to otitis media has been reported. However, the specific mechanisms involved are not clearly understood. The aim of this study is to investigate the expression of macrophage migration inhibitory factor and CD74 in the middle ear and inner ear in lipopolysaccharide-induced otitis media.Method: BALB/c mice received a transtympanic injection of either lipopolysaccharide or phosphate-buffered saline (PBS). The mice were sacrificed 24 h after injection, and temporal bones were processed for polymerase chain reaction (PCR) analysis, histologic examination, and immunohistochemistry.Results: PCR examination revealed that the lipopolysaccharide-injected mice showed a significant up-regulation of macrophage migration inhibitory factor in both the middle ear and inner ear as compared with the PBS-injected control mice. The immunohistochemical study showed positive reactions for macrophage migration inhibitory factor and CD74 in infiltrating inflammatory cells, middle ear mucosa, and inner ear in the lipopolysaccharide-injected mice.

Diagnosis and prevalence of ovine pulmonary adenocarcinoma in lung tissues of naturally infected farm sheep

Aim:This study was aimed to detect ovine pulmonary adenocarcinoma (OPA) in sheep flocks affected with pulmonary disorders at organized farm.Materials and Methods:A total of 75 sheep died naturally were thoroughly examined for the lesions of OPA during necropsy. Tissue sections from affected portion of the lungs from each animal were collected aseptically and divided into two parts; one each for polymerase chain reaction (PCR) and another for histopathology.Results:On PCR examination of lung tissues, six sheep (8%) were found to be positive for JSRV. Two of them were 3-6 months of age and did not show clinical signs/gross lesions of OPA. Four adult sheep positive on PCR revealed characteristic lesions of OPA on gross and histopathological examination.Conclusion:In the absence of known specific antibody response to the infection with JSRV, there is no diagnostic serological test available. The PCR assay employed in this study on lung tissues, using primers based on the U3 region of the viral long terminal repeat for JSRV would be helpful in the screening of preclinical and clinical cases of OPA in sheep.

English

Porcine circovirus type 2 (PCV2) is an important infection factor causing post weaning multisystemic wasting syndrome (PMWS). The amount of PCV2 viral load may mainly give rise to clinical symptom and pathological lesion of PMWS. In order to investigate the relationship between PCV2 viral load and the lesion severity of lymph tissues, and search further for some clues of pathologic diagnosis of PMWS, thirty pig cases affected with PCV2 and aged 30i½ž90 days old were collected from swine farms and their lymph tissues were treated by quantitative Real Time PCR, immunohistochemistry (IHC) and histopathology examination. Four various groups were classified according to their evaluation scores for lesion severity, and it is shown that the higher the score for pathological lesion of lymph tissues, the more there is viral load in tonsil. Especially, the amount of PCV2 DNA in group one was 1/1000 lower than other three groups. It is supposed that group one is considered as subclinical case and the other three groups as clinical PMWS cases. Furthermore, it is likely presumed whether PCV2 infection is subclinical or clinical PMWS case can be helpfully diagnosed by these criteria. Key words: Correlation, PCV2 load, Real Time polymerase chain reaction (PCR), immunohistochemistry (IHC).

Mycobacterium tuberculosis Contaminant Risk on Bone Marrow Aspiration Material from Iliac Bone Patients with Active Tuberculous Spondylitis.

There was a concern on Mycobacterium tuberculosis spreading to the bone marrow, when it was applied on tuberculous spine infection. This research aimed to study the probability of using autologous bone marrow as a source of mesenchymal stem cell for patients with tuberculous spondylitis. As many as nine patients with tuberculous spondylitis were used as samples. During the procedure, the vertebral lesion material and iliac bone marrow aspirates were obtained for acid fast staining, bacteria culture, and PCR (polymerase chain reaction) tests for Mycobacterium tuberculosis at the Clinical Microbiology Laboratory of Faculty of Medicine Universitas Indonesia. This research showed that there was a relationship between diagnostic confirmation of tuberculous spondylitis based on the PCR test and bacterial culture on the solid vertebral lesion material with the PCR test and bacterial culture from the bone marrow aspirates. If the diagnostic confirmation concluded positive results, then there was a higher probability that there would be a positive result for the bone marrow aspirates, so that it was not recommended to use autologous bone marrow as a source of mesenchymal stem cell for patients with tuberculous spondylitis unless the PCR and culture examination of the bone marrow showed a negative result.

Identification of Trypanosoma species infecting equine in Egypt using Polymerase Chain Reaction (PCR)

This study was carried out to identify the Trypanosoma species in symptomatic and asymptomatic horses and donkeys in Egypt by using polymerase chain reaction (PCR). Blood samples were collected from 80 horses and 37 donkeys from different farms and private owners at Cairo and Giza governorates. Thin blood smears were prepared from each sample and examined microscopically. Four samples for Trypanosoma species were positive by using microscopic examination of stained blood smears from horses. While, all samples collected from donkeys were negative for Trypanosoma species infection. Microscopic examination of stained thin blood smears revealed that the parasite were monomorphic thin trypomastigote parasite, long free flagellum and thin posterior extremity with subterminal small kinetoplast and length ranges from 18-34 micron (mean 26). The four positive samples from horses were subjected to PCR using three sets of Trypanosoma species specific primers (T. brucei group, T. evansi and T. equiperdium). Three of the 4 positive samples showed symptoms of Trypanosoma infection and one case asymptomatic. PCR assay. identified that the 4 samples were positive for T. brucei group and Trypanosoma evansi species using Trypanosoma brucei TBR 1/TBR 2 specific primer at 164 bp and T. evansi EVA 1 /2 specific primer at 138 bp While, the same four samples showed negative results using T. equiperdium Nade 5 specific primer at 395 bp. Six negative samples from horses and donkeys examined microscopically were subjected to PCR examination. These samples were negative for the three species of Trypanosoma. From this it can be concluded that, PCR assay was a highly sensitive diagnostic technique, for exact discrimination between the species of Trypanosoma infection (T. brucei group, T. evansi and T. equiperdium) in equines. Therefore, Trypanosoma infection in horses in Egypt is most probably due to T. evansi.

Halal authenticity of gelatin using species-specific PCR

Consumption of food products derived from porcine sources is strictly prohibited in Islam. Gelatin, mostly derived from bovine and porcine sources, has many applications in the food and pharmaceutical industries. To ensure that food products comply with halal regulations, development of valid and reliable analytical methods is very much required. In this study, a species-specific polymerase chain reaction (PCR) assay using conserved regions of mitochondrial DNA (cytochrome b gene) was performed to evaluate the halal authenticity of gelatin. After isolation of DNA from gelatin powders with known origin, conventional PCR using species-specific primers was carried out on the extracted DNA. The amplified expected PCR products of 212 and 271bp were observed for porcine and bovine gelatin, respectively. The sensitivity of the method was tested on binary gelatin mixtures containing 0.1%, 1%, 10%, and 100% (w/w) of porcine gelatin within bovine gelatin and vice versa. Although most of the DNA is degraded due to the severe processing steps of gelatin production, the minimum level of 0.1% w/w of both porcine and bovine gelatin was detected. Moreover, eight food products labeled as containing bovine gelatin and eight capsule shells were subjected to PCR examination. The results showed that all samples contained bovine gelatin, and the absence of porcine gelatin was verified. This method of species authenticity is very useful to verify whether gelatin and gelatin-containing food products are derived from halal ingredients.

Genotypic Speciation of Four Plasmodium among Human Immunodeficiency Virus Positive Individuals Attending HIV Clinics in Abakaliki, South-Eastern Nigeria

A total of 150 blood samples were collected from Human immunodeficiency virus (HIV) positive patients who visited selected hospitals in Ebonyi State. The subjects were made up of 57 males and 93 female patients. The blood samples were screened for the presence of four human malaria parasites using parasitological examination of blood stained films and Polymerase Chain Reaction (PCR). Of the 150 positive individuals, 75(50%) blood samples were positive for malaria ( P. falciparum ). The comparison of blood films microscopy and PCR results were evaluated thus, 88 malaria positive cases recorded a prevalence of 58.68% for malaria parasites by PCR analysis while the overall prevalence of malaria infections by microscopy gave 50% prevalence. However, there were a number of disagreements in the identification of Plasmodium species by these two methods. Ten (6.67%) subjects were identified by PCR to be infected by P. malariae while blood film microscopy yielded 4(2.67%). Microscopy gave 70(46.67%) malaria positive cases of P. falciparum while PCR analysis yielded 75(50%). Two percent of the subjects screened were determined to be a mixed infection of P.falciparum and P. malariae by PCR while microscopy result revealed 0.67% prevalence. Therefore, PCR examination proves more sensitive than the parasitological technique used in malaria parasite studies.

Comparison of Tuberculin Skin Test, IFN-γ Assay, Real Time PCR and Lateral Flow Rapid Test in Diagnosis of Field Outbreaks of Bovine Tuberculosis

Bovine tuberculosis is an important zoonotic disease transmitted by direct contact, respiratory pathway, ingestion of unpasteurised milk and milk product, raw or undercooked meat. Tuberculosis can be difficult to diagnose based only on the clinical signs. Tuberculosis is usually diagnosed in the field with the tuberculin skin test. Sputum and other body fluids may be collected for microbiological examination. Polymerase chain reaction (PCR) methods have also been described. Diagnostic blood tests include the lymphocyte proliferation assay, the interferon gamma (IFN-γ) assay, and enzyme-linked immunosorbent assays (ELISA). In this study a total of 50 animals were tested by using tuberculin skin test (TST), lateral flow rapid test, IFN-γ assay and real time PCR. The animals were selected randomly among 178 cattle in dairy farms with the aged between 3-5 years and suspected of having tuberculosis. Forty five cattle were positive out of 50 for TST while 31 for reactive by the IFN-γ assay and 28 for rapid test and 9 for real time PCR. The purpose behind such variable as age was to compare sensitivity of tuberculin skin test, the IFN-γ assay and TB lateral flow rapid test and real time PCR examination for the diagnosis of field outbreaks of bovine tuberculosis in Turkey.

Gonorrhoea Infection Prevalence in Human Immunodeficiency Virus Positive Patients Based on Polymerase Chain Reaction Examination

Objective: To determine the prevalence of gonorrhoea infection in male and female HIV positive patients based on polymerase chain reaction (PCR) examination at the Teratai Clinic, Dr. Hasan Sadikin General Hospital Bandung. Methods: This study was conducted in July, 2012 at the Teratai Clinic, while the PCR examination was performed at the Molecular Biology Laboratory of Microbiology Department, Faculty of Medicine, Universitas Padjadjaran, Bandung. This was a cross-sectional observational study. The subjects were 81 HIV positive patients who were taken in consecutive admission. They underwent history taking and physical examination. Samples were taken from urethral swab in males and cervical swab in females for PCR examination. Results: The PCR examination result was positive for gonorrhea in 36% subjects. From all male subjects participating in the study, 37% were positive while 33% of the female subjects were also positive for gonorrhea. Conclusions: The prevalence of gonorrhoea infection in male and female HIV positive patients at the Teratai Clinic, Dr. Hasan Sadikin General Hospital Bandung is quite high, i.e 37% and 33%, respectively. Keywords: Gonorrhoea, human immunodeficiency virus, polymerase chain reaction, prevalence DOI : 10.15850/ijihs.v2n1.270

Clinical, immunohistochemical, Western blot, and genetic analysis in dystrophinopathy

Dystrophin-deficient muscular dystrophies (dystrophinopathies) are the most common form of muscular dystrophy, with variable clinical phenotypes ranging from the severe Duchenne (DMD) to the milder Becker (BMD) forms. In this study, we investigated the relationship between clinical characteristics, findings at immunohistochemistry (IHC) and Western blot, and the pattern of exon deletions in 24 male patients with dystrophinopathies. We retrospectively reviewed findings from clinical and laboratory examinations, IHC for dystrophin of muscle biopsy tissue, Western blot analysis, and multiplex polymerase chain reaction (PCR) examination of genomic DNA. All tests were performed in every patient. PCR examination revealed exon deletions in 13 patients (54.2%). At Western blot analysis, 15 patients (62.5%) were negative at all three dystrophin domains. Most of these patients had a clinical presentation consistent with the DMD phenotype. Nine (37.5%) others were weakly positive at one or more domains. Most of these patients presented clinically as BMD phenotype. One patient whose clinical presentation was consistent with BMD phenotype had normal findings at IHC and was weakly positive at all three domains on Western blot analysis; however, with the exception of this patient, the findings at IHC and Western blot were consistent for individual patients. Based on these findings, we conclude that Western blot analysis appears useful for confirmation of dystrophinopathy in BMD patients with normal staining on IHC. Exon deletion analysis by multiplex PCR using peripheral blood is also a simple and useful test for the diagnosis of dystrophinopathy, although it has limited sensitivity.

Use of a Comprehensive Polymerase Chain Reaction System for Diagnosis of Ocular Infectious Diseases

To measure the genomic DNA of ocular infectious pathogens in ocular fluids and to analyze the clinical relevance of these pathogens in uveitis and endophthalmitis. Prospective clinical case series. A total of 500 patients with infectious uveitis and endophthalmitis were examined at Tokyo Medical and Dental University, Tokyo Medical University, Kyushu University, Osaka University, and Kyoto Prefectural University, all in Japan. Genomic DNA of bacteria, fungi, parasites, and viruses in collected intraocular samples were examined by comprehensive polymerase chain reaction (PCR). Samples were analyzed first by multiplex PCR and quantitative real-time PCR for human herpes viruses (HHVs) 1 through 8 and toxoplasma. Subsequently, samples were examined by broad-range real-time PCR for bacterial 16S and fungal 18S/28S ribosomal DNA (rDNA). Infectious uveitis and endophthalmitis diagnoses were obtained when using the PCR system. Calculations of the positivity and the diagnostic parameters such as sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) also were evaluated. In all of the tested infectious uveitis and endophthalmitis patients, either herpes simplex virus type 1 (n = 18), herpes simplex virus type 2 (n = 4), varicella-zoster virus (n = 55), Epstein-Barr virus (n = 17), cytomegalovirus (n = 68), HHV type 6 (n = 2), toxoplasma (n = 6), bacterial 16S (n = 33), or fungal 18S/28S (n = 11) genome was detected. Neither HHV type 7 nor HHV type 8 DNA was detected in any of the samples. Of the 21 false-negative results found during the PCR analyses, 12 cases were negative for patients clinically suspected of having bacterial endophthalmitis. Conversely, false-positive results for the comprehensive PCR examinations occurred in only 3 cases that subsequently were found to have bacterial 16S rDNA. Diagnostic parameters for the sensitivity, specificity, PPV, and NPV of our PCR examinations were 91.3%, 98.8%, 98.6%, and 92.4%, respectively. Use of our comprehensive PCR assay to examine ocular samples in patients with endophthalmitis and uveitis seems to be clinically useful for detecting infectious antigen DNA. Thus, this PCR method is a reliable tool for both diagnosing ocular disorders and further screening of patients for intraocular infections. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

A Case of Delayed Sinusoidal Obstruction Syndrome (SOS) Administered with Recombinant Soluble Thrombomodulin (rTM) and Peritoneovenous Shunt

The subject is a 45-year old female who visited the hospital due to irregular vaginal bleeding. Her white blood cell count was 170400/ uL and LDH level was 2099 iU/L. The subject was diagnosed with acute myeloid leukemia type M2 by bone marrow examination. The chromosomes were 46, XX, add(3)(p21), add(8)(q22), and add(21)(q22). The PCR (Polymerase Chain Reaction) examination confirmed that the positive AML1/ETO. Although one course of idarubicin hydrochloride (IDR: 12 mg/m2 for three days) plus cytarabine (AraC: 100 mg/m2 for seven days) was conducted as remission induction therapy, remission was not achieved. Since the bone marrow examination also observed meningeal infiltration, a second course of IDR plus AraC was combined with intraspinal administration of antitumor drugs, aiming for reremission induction. Although the meningeal infiltration disappeared, non-remission status remained. We then conducted two courses of FLAG-M (AraC 2 g/m2 twice a day for four days, fludarabine 15 g/m2 twice a day for four days, mitoxantrone 10 mg/ m2 for three days, and granulocyte colony-stimulating factor: G-CSF 75 μg) and one course of MEC (AraC 1 g/m2 for six days, etoposide 80 g/m2 for six days, and mitoxantrone 6 mg/ m2 for six days). These regimens did not lead to a complete remission.

Rapid Identification ofStaphylococcus aureus: FISH Versus PCR Methods

Background: Staphylococcus aureus (S aureus) is the most important pathogen in the genus Staphylococcus. The ability to rapidly and accurately distinguish between S aureus and non–S aureus bacteria (coagulase-negative Staphylococcus species [CoNS]) is essential for the appropriate therapeutic use of antibiotics and timely intervention for infection control. Several methods have been reported as being effective in the rapid identification of S aureus, among which molecular biological methods have the most potential. Methods: In this study, 2 molecular techniques, fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR), were compared in 1300 clinical specimens. After smear testing, specimens containing gram-positive cocci in clusters were submitted for investigation. These specimens had been subjected to FISH analysis and PCR examination. Overall, we gathered statistics on 131 specimens that had been determined to be members of the Staphylococcus genus. We compared the effectiveness, efficiency, and costliness of the 2 methods. Results: Comparing the culture determination methods revealed that the identification sensitivity, specificity, and positive and negative predictive values were 100%, 100%, 100% and 100%, respectively, for the FISH method and 98.5%, 100%, 100% and 98.5%, respectively, for the PCR method. The FISH method took approximately 3 hours to complete per sample, whereas PCR took approximately 4 hours. S aureus was differentiated from CoNS via the FISH method 1 hour faster than via PCR identification. Conclusion: Although the FISH and PCR methods both allow for the rapid and reliable identification of S aureus in clinical specimens, FISH is more appropriate for testing a few specimens, whereas PCR is more appropriate for testing a large number of specimens at a lower cost.

Field Evaluation of a PCR Test for Schistosoma japonicum Egg Detection in Low-Prevalence Regions of China

Sensitive Schistosoma japonicum detection methods are needed to progress from schistosomiasis control to elimination. The sensitivity of the Kato-Katz thick smear and miracidium hatching tests decrease with infection intensity and serological tests cannot always identify current infections. We evaluated a fecal polymerase chain reaction (PCR) assay to detect S. japonicum infection in 106 humans and 8 bovines in China. PCR was highly sensitive, detecting S. japonicum DNA at 0.5 eggs/g of stool. Comparing PCR examination of a single stool sample to the miracidium hatching test using three consecutive stool samples, more humans were hatching test positive (20%) than PCR positive (15%). However, two individuals were PCR positive in a village where no infections were detected by coprological methods. The sensitivity of PCR makes it a promising tool for schistosomiasis diagnostics and screening, although egg shedding variability and stool sample size present challenges for any detection method in low-transmission areas.

Comparison of diagnostic tools with multiplex polymerase chain reaction for pediatric lower respiratory tract infection: A single center study

Acute respiratory tract infections are a leading cause of morbidity and mortality in children worldwide. Most have a viral etiology, with pneumococcus as an important pathogen. This single-center study compared the use of conventional diagnostic tools and two multiplex polymerase chain reaction (PCR) examinations for determining pathogens in lower respiratory tract infections (LRTIs) among children aged <5 years. From July to October 2010, 45 patients aged 2 months to 60 months and diagnosed as having LRTIs were enrolled. Their nasopharyngeal aspirates were evaluated through viral culture and two multiplex PCR examinations. The patients' clinical course, symptoms, signs, and laboratory findings were recorded and analyzed. Among the 45 patients, 38 (84.4%) had detectable pathogens. Conventional viral and blood cultures had 35.6% positive rate, which increased to 51.1% when the quick antigen tests (Influenza A+B test and respiratory syncytial virus) and urine pneumococcal antigen test were combined. The positive rate further increased to 84.4% when the two multiplex PCR methods were combined. Twelve patients had co-infection, including 10 detected by the multiplex PCR methods. The co-infection rate was 26.7% (12/45). Most LRTIs in children have a viral etiology. Multiplex PCR tests are rapid assays that can increase the diagnostic yield rate and detect slow-growing viruses and can detect more pathogens than conventional viral culture to enable, thereby helping clinicians to provide appropriate and timely treatment.

Prevalence ofCoxiella burnetiiin Hungary: Screening of Dairy Cows, Sheep, Commercial Milk Samples, and Ticks

Q fever is an important zoonotic disease caused by Coxiella burnetii. There are few reliable data about C. burnetii infection available. The aim of this study was to assess the importance and potential infectious sources of Q fever in Hungary. A total of 215 milk samples (10 individual samples from each herd and 1 bulk tank milk sample from each cattle herd), and 400 serum samples (20 from each herd) were tested from 15 dairy cattle herds and 5 sheep flocks located in different parts of Hungary. The study found 19.3% (58/300) and 38.0% (57/150) seropositivity in cattle, and 0% (0/100) and 6.0% (3/50) seropositivity in sheep, by complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA), respectively. C. burnetii DNA was detected by IS1111 element-based TaqMan real-time polymerase chain reaction (PCR) in 8.7% (13/150) of individual dairy cow milk samples, 4.0% (2/50) of individual sheep milk samples, and 66.7% (10/15) of dairy bulk tank milk samples. Samples taken from nine different commercially-available pasteurized cow milk products from different Hungarian producers were also tested for the presence of C. burnetii DNA, and eight of these samples were found to be positive (88.9%). The real-time PCR examination of 5402 ixodid ticks collected from different parts of the country yielded negative results. Knowledge of the true prevalence of Q fever is crucial for policymakers involved in evidence-based decision making.

NELL1 Promotes Bone Regeneration in Polyethylene Particle-Induced Osteolysis

We investigated the therapeutic effects of a craniosynostosis-associated molecule, NEL-like molecule-1 (NELL1; NEL [a protein strongly expressed in neural tissue encoding the epidermal growth factor-like domain]), on osteolysis induced by polyethylene (PE)-particle debris. We used a murine calvarial osteolysis model with in vivo adenovirus (Ad)-mediated gene transfer. In total, 76 female Balb/c mice were randomly assigned to four groups for treatment 1 day postoperation: SHAM (injected with 0.1 mL saline without implantation of particles); PE control (injected with 0.1 mL saline after implantation of particles); PE+(Ad-GFP-NELL1) (injected with 0.1 mL Ad-GFP-NELL1 in saline after implantation of particles); and PE+(Ad-GFP) group (injected with 0.1 mL Ad-GFP in saline after implantation of particles). Green fluorescent protein (GFP) and NELL1 delivery in vivo after the injection were validated by optical imaging at 10 day postop, and then, all mice were sacrificed for analysis by three-dimensional (3D) microcomputed tomography (micro-CT), real-time polymerase chain reaction (PCR), histology, and biomechanical testing. Exogenous NELL1 and GFP were expressed in the osteolysis area for at least 9 days after the Ad-GFP-NELL1 injection. Serial 3D micro-CT images and testing of bone volume, bone mineral density, trabecular thickness, bone surface density, and connectivity density revealed that the new bone promoted with the Ad-GFP-NELL1 injection could almost compensate the PE-induced osteolysis and regenerate significantly better than with the Ad-GFP treatment. The expression of osteopontin (OPN) was significantly higher with Ad-GFP-NELL1 transduction among all the samples. Real-time PCR examination confirmed the augmented expression of OPN, Runx-2, and receptor activator of nuclear factor-kappa B ligand (RANKL). The elastic modulus was significantly greater with Ad-GFP-NELL1 than with the PE and/or Ad-GFP group (p<0.01). We found no transgene-associated toxic effects. Ad-GFP-NELL1 gene transfer effectively reversed the calvarial osteolysis and could be considered a new treatment for osteolysis through promoting bone regeneration.