جستجوی مقالات مرتبط با کلیدواژه « Nested-PCR » در نشریات گروه « دامپزشکی »

Anaplasma sp. is a blood protozoon that causes economic damage to the livestock industry. Therefore, studying this disease’s epidemiology and distribution pattern in different regions is essential. 

This study aimed to investigate the variety of infections of the Anaplasma sp. in the sheep population of Khuzestan Province in Iran.

A total of 200 sheep blood samples were randomly collected and examined using specific nested polymerase chain reaction (nPCR) based on the 16S rRNA gene. 

The prevalence of Anaplasma phagocytophilum was 17%, and infected sheep had no clinical signs. The effective risk factors in the spread of infection in Khuzestan Province include sheep aged 3-5 years, low sanitation, high-density farms, use of acaricides in the field, and hot season (P≤0.05). There was no significant association between the occurrence of A. phagocytophilum infection and variables of altitude, farm type, vectors, distance from other farms, and sex.

Since the infection often has no clinical symptoms, identifying the risk factors and epidemiology is essential to develop control and prevention planning.

Anaplasmosis is a tick-borne disease with worldwide distribution, affecting ruminants, equines, carnivores, and humans. This study aimed to investigate Anaplasma phagocytophilum (A. phagocytophilum) in horses from Ardabil province and Anaplasma ovis (A. ovis) in small ruminants from East Azerbaijan province using the Nested PCR method. Blood samples were collected from the jugular vein of 100 healthy horses in the Ardabil province and 156 healthy sheep and goats (116 sheep and 40 goats) in the East Azerbaijan Province during the spring and summer seasons of 2016 in northwest Iran. The gathered blood samples were stored at -20°C until the molecular experiments were conducted. Nested PCR was employed to detect A. phagocytophilum in horses and A. ovis in small ruminants using extracted DNA and amplifying 16S rRNA and msp4 genes. The Chi-square test of independence was used to determine the relationship between Anaplasma spp., infection, and independent variables, including age, gender, animal species, and sampling location. None of the 100 samples collected from horses in the Ardabil province were positive for A. phagocytophilum. In the East Azerbaijan province, 11 out of the 156 (7.05%) blood samples collected from sheep and goats tested positive for A. ovis. In addition, A. ovis infection was not significantly related to the independent variables. Phylogenetic analysis showed that the sequence obtained in the present study (MH790273) has 100% homology with the sequence obtained from sheep infected with Anaplasma in Ahvaz (JQ621903.1). The study’s findings can aid in preventing and controlling anaplasmosis in farm animals in northwestern Iran.

Paratuberculosis (Johne's disease) is a chronic granulomatous small intestine disease caused by MAP. Diagnosing and isolating infected animals is the most important measure for controlling the disease. Therefore, this study aimed to molecularly identify mycobacterium isolated from ELISA-positive cows with Johne's disease by nested PCR from the samples from Markazi Province, Iran. For this purpose, 2938 samples were decontaminated and then cultured on the Herrold egg culture medium containing mycobactin and no mycobactin. After DNA extraction, PCR for 16S rRNA was first performed, followed by nested PCR on positive samples. Of 2938 samples, 87 were positive, and 26 were suspected. All positive isolates were observed in Ziehl-Neelsen staining in microscopic expansion. A 543-bp band was observed in 26 tested samples and mycobacterium strains in PCR for 16S rRNA, indicating the presence of mycobacterium in the above samples. Nested PCR was performed for all isolates and positive and negative control strains. A 398-bp band was obtained in the first stage, and a 298-bp fragment was obtained in the second stage, indicating the presence of MAP in the samples. Accordingly, nested PCR is suggested as a proper method for the quick and definitive diagnosis of disease cases.

Mycobacterium avium complex (MAC) is connected to human immunosuppressive diseases, including HIV-AIDS, and may pose a zoonotic threat. MAC causes lymphadenopathy in children, respiratory infection in adults, and generalized infection in immunocompromised individuals. Infection with nontuberculous Mycobacteria (NTM) in humans is now primarily brought on by MAC. Recently, MAC members have emerged as pathogenic organisms for animals and humans. While dogs are generally resistant to mycobacterial infections, there have been some cases of infection that result in systemic or disseminated diseases. The organisms can be transmitted to dogs through oral contact, and their faeces can be a possible source of infection for dog owners. It is important to note that this ailment is zoonotic, especially if infected pet dogs are in prolonged contact with their humans.

The study was planned to demonstrate the occurrence of MAC organisms and other Mycobacteria in dogs associated with lymphadenopathy cases with special emphasis on lymphadenitis.

A total of 123 samples (100 lymph node aspirates, 15 lymph node tissues, and 8 blood samples) from 83 dogs suspected of lymphadenitis accompanied by gastroenteritis, chronic skin infections, immunosuppression, chronic pulmonary diseases, and other chronic undiagnosed diseases were studied. The samples were processed for cytological and microscopic examination by Ziehl-Neelsen staining. Following the decontamination procedure, the aspiration and lymph node tissue samples were inoculated into Middlebrook 7H11 media for up to 8 weeks. The aspirated material was also directly used for molecular detection by triplex-nested polymerase chain reaction (nPCR) assay.

A cytological study revealed pyogranulomatous inflammation of the lymph node tissue. Impression smears from lymph node tissues displayed the presence of acid-fast organisms. Out of 83 cases of dogs, 8 were found to be positive for Mycobacterium spp. Among those 8 positive cases, 3 were confirmed to belong to MAC, and 5 belonged to the Mycobacterium tuberculosis complex (MTB complex).

MAC and MTB are the underestimated bacteria that could be the causative agents of lymphadenitis in animals.

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.

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.

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.

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).

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.

Cutaneous leishmaniasis (CL) is a vector-borne disease widely distributed in tropical and subtropical areas of North and South America, Europe, Asia, and Africa. Considering the increasing number of CL cases in recent years and the fact that no study has been conducted to identify CL fauna and vectors in Alborz province, this study was carried out to identify sand flies and CL vectors in this region. Sand flies were collected from August to October 2021 from plain and mountainous indoor and outdoor areas of the region using sticky paper traps and were detected morphologically. DNA was extracted from the midguts of female sand flies. In this study, 1157 sand flies were collected and identified. The number of sand flies caught from indoor and outdoor places was 367 (31.72%) and 790 (68.28%), respectively. Overall, six species of flies were of the genus Phlebotomus (Raynal, 1937), including Phlebotomus papatasi (P. papatasi, 695 [60.07%]; Scopoli, 1786), P. kandelakii (13 [1.12%]; Shchurenkova, 1926), P. sergenti (232 [20.05%]; Parrot, 1917), P. major (14 [1.21%]; Annandale, 1910), P. caucasicus (4 [0.35%]; Marzinowsky, 1917), P. alexandri (18 [1.56%]; Alexandri Sinton, 1920), and four were of the genus Sergentomyia (Artemiev, 1978), including Sergentomyia tiberiadis (109 [9.42%]; Adler, Theodor & Lourie, 1930), Sergentomyia baghdadis (53 [4.58%]), Sergentomyia sintoni (14 [1.21%]; Sintoni Pringle, 1933), Sergentomyia clydei  (5 [0.43%]). P. papatasi spp. were dominant in indoor and outdoor places, with a prevalence of 695 (60.07%). The Leishmania major (L. major) gene was identified in five samples of P. papatasi spp. This suggests that P. papatasi is the potential vector spp. in the study area. Moreover, L. major was confirmed as the aetiological agent of CL cases in Alborz province. The identification of vectors and parasite spp. is very important for the treatment and operational planning of disease vectors.

Toxoplasmosis is one of the most widespread zoonotic diseases, especially in warm and humid areas, and affects all mammals, including humans and many herbivores and carnivores. The present study investigated the Toxoplasma gondii (T. gondii) parasite in tortoises for the first time in Iraq using PCR technology. A total of 28 tortoises/Testudo graeca (T. graeca) were collected between October 2018 and March 2019 from the study stations and then sent to the Animal House, which belongs to the Department of Biology, Faculty of Education, University of AL-Qadisiyah, Iraq, to perform the dissection. The body cavity was opened, and all organs were removed. The tortoises’ liver, heart, and brain were removed and kept at -20ºC until use. Afterward, the samples were subjected to DNA extraction. The Nested-PCR technique was implemented using two pairs of primers, and then the PCR products were analyzed using 1.5% agarose gel electrophoresis. The amplification of the gene during the first cycle indicated that 10 samples gave positive results with a total percentage of (11.9%), including five liver samples, three heart samples, and two brain samples (17.85%, 10.71%, and 7.14%, respectively). On the other hand, during the second cycle of the reaction, the amplification of the gene was obtained in seven samples (8.33%). The highest percentage of the presence of the gene was recorded in the tortoises’ liver (14.28%) and the lowest in their brain (3.57%). This study is among the first to investigate the molecular detection of T. gondii in wild tortoises (T. graeca) in Iraq. The findings imply that tortoises have a role in transmitting T. gondii and are believed to acquire infection by feeding on small invertebrate animals or plants contaminated with the oocysts of the parasite.

The selection of suitable cell cultures for use in the biological industry as well as research and diagnostic studies is critical. One of the factors affecting cell culture that can affect the results of studies is the contamination with viral agents. Therefore, efforts to preserve the health of cell cultures from contamination sound logical, and the use of virus-free cells is vital in research and diagnostic studies as well as in the manufacturing industries. For this purpose, it is crucial to use fast and correct diagnostic methods to detect the presence of critical viral contaminants in cells. Moreover, the use of susceptible diagnostic methods is also doubly important, especially in the case of contaminants that may remain hidden. Therefore, in this study, the BHK-21C5 cell line, one of the most widely used cells in the production and quality control of biological products and virological studies, was examined in terms of contamination with the most important viruses such as BVD and BLV. Detection of possible contaminants by using two-step RT-PCR to detect the 5ʹ UTR portion of the BVD virus. Moreover, Nested PCR was carried out to detect the BLV virus using the gp51 env gene region. Also, an Flk cell line and Hela cell line that were consistently contaminated with the BLV virus were used as positive controls in Nested PCR. Due to the absence of bands in the BHK-21C5 cell column and the bandwidth observed in the positive control column (BVDV) in the range of 283 bp, non-contaminating of the cell clone with the BVD virus was proved. Also, no band was observed in well related to BHK-21C5 cell, and no cell clone contamination with BLV was confirmed, and in wells related to the positive controls (Flk and Hela cells), the bands have seen in the range 444bp. So, the results showed that no obvious or covert viral contamination effect was observed in the cell clone studied. Hence, the use of this cell line seems unobstructed in the quality control and production of biological products and research and diagnostic studies.

Anaplasmosis is caused by an obligate intracellular, gram-negative microorganism, which be-longs to the family Anaplasmatacea and can be transmitted by ticks and other arthropods.

The present study aimed to investigate the status of Anaplasma spp. infection by microscopy and molecular methods in dromedary camels in Bushehr province, Iran.

A total of 139 blood samples were collected from dromedary camels in Bushehr province. Giemsa staining and nested-polymerase chain reaction (PCR) were conducted to detect Anaplasma infection in the drome-dary camels.

We found that 27 (19.4%) out of the total 139 blood samples were suspected for the presence of Ana-plasma spp. by morphological study. The PCR and nested-PCR sequencing results showed 111 (80%) and 134 (96%) samples positive for Anaplasma spp. and BLAST search in NCBI GenBank presented 100% identity with Candidatus Anaplasma camelii.

The molecular results presented the high frequency of Candidatus Anaplasma camelii in camels, in Bushehr city.

Chlamydophila abortus is one of the major causes of pregnancy failure (abortion) in sheep and goats in many countries. In the present study, milk samples from sheep and goat herds of West Azerbaijan, Iran were examined for C. abortus using PCR and nucleotide sequencing. A total number of 360 milk samples were randomly collected from sheep (n=180) and goats (n=180) of three different regions of West Azerbaijan province during 2018. DNA was isolated from samples and the nested-PCR was employed targeting the 16S rRNA gene for detection of Chlamydia spp. The omp gene was amplified and sequenced for the characterization of detected C. abortus.The results showed that 8.61% (95% CI: 6.13%–11.96%) of the examined samples (11.67% sheep and 5.56% goat milk samples) were positive for C. abortus. The frequency of positive samples in the central region was significantly higher than in other regions. Positive samples for C. abortus from animals with a history of abortion were significantly higher than those without a history of abortion. Positive samples in autumn were significantly higher than the other seasons and also, in animals more than four years old were significantly higher than other age groups. Sheep infection was significantly higher than the goats. Phylogenetic analysis based on the helicase gene showed that two sequenced isolates clustered closely with the other C. abortus isolates reported in the GenBank. In conclusion, small ruminants in West Azerbaijan province were contaminated with C. abortus and they could shed this organism into the milk.

Brucellosis is recognized as a zoonotic disease with high morbidity in the absence of treatment. The primary diagnosis of brucellosis can be effective in the achievement of satisfying treatment results and prevention of chronic infections. The present study aimed to compare the efficiency of conventional microbiological and serological approaches with nested Polymerase chain reaction (nested PCR) for rapid diagnosis of human brucellosis. A total of 120 subjects with symptoms of brucellosis were included in the study. The sensitivity and specificity of nested PCR for the detection of Brucella bacteria were compared with serological and blood culture methods. Out of 120 patients enrolled, brucellosis was detected in 73 (60.83%) cases based on serological tests with a blood culture confirmation in 8.33% of participants. Based on the obtained results, 55% of cases were positive in serum agglutination test (SAT≥1:160), and Coombs (C-SAT≥1:160) tests. Furthermore, seven negative SAT cases were positive in C-SAT as evidence of chronic brucellosis. The results of the 2-mercaptoethanol (2-ME) ≥ 1:80 test were negative in six SAT-positive cases. Based on nested PCR results, 68.18% and 56.06% SAT positive samples were also detected by blood nested PCR and serum nested PCR, respectively. The sensitivity of blood nested PCR was significantly more than serum nested PCR, SAT≥1:160, and blood culture (P<0.001). Moreover, the specificity of blood and serum nested PCR was obtained at 100%, compared to blood culture and SAT≥ 1:160. In the present study, the nested PCR was able to identify chronic brucellosis in SAT negative patients. As evidenced by the obtained results, the nested PCR showed higher efficiency for rapid diagnosis of human brucellosis, as compared to the blood culture method. Furthermore, the findings pointed to the high performance of nested PCR for rapid diagnosis of both chronic and acute brucellosis.

Bovine leukemiavirus (BLV) is one of the most important carcinogenic viruses genetically related to the human T-cell lymphotropic viruses (HTLV-1 and HTLV-2). The virus infects type B lymphocytes and creates lymph glands tumors. Recently, the association between the presence of this virus and breast cancer has been addressed in humans. Here, we studied the prevalence of BLV in the samples of raw milk of native Iranian and Iranian-foreign cows in traditional, semi-industrial and industrial dairy farms in rural and urban areas of Zanjan province. Raw milk samples of cows were collected manually in sterile tubes. The samples were tested by nested-PCR method. Forty samples (9.93%) out of 403 samples showed BLV contamination. In this study, nested-PCR was successfully applied to determine the level of contamination in raw milk samples from cows infected with BLV. Furthermore, a relatively high rate of BLV infection was found in dairy cows in Zanjan province, northwestern of Iran.

Members of the genus Mycoplasma are known as pathogens causing respiratory disease in cattle world-wide.

The present study aimed to investigate mycoplasmal infection in the lung tissue of cattle slaughtered in Hamadan industrial abattoir, Iran, using molecular and histopathological methods.

A total of 108 tissue samples were collected from the cranioventral parts of the cattle lung during March 2015-February 2016. The specimens were subjected to a polymerase chain reaction (PCR) and histopathological examinations. The PCR-positive samples were tested subsequently for Mycoplasma bovis and Mycoplasma dispar using nested PCR assay.

Nine (8.33%) samples contained the DNA of genus Mycoplasma, among which, five and one showed the DNA sequences of M. bovis and M. dispar, respectively. Pathological changes, such as caseonecrotic lesions, interstitial pneumonia, lobar bronchopneumonia, and bronchial atelectasis were observed in 24 (22.22%) tissue samples. All the PCR-positive lungs demonstrated at least one pathological manifestation. However, not every pathognomonic tissue changes were concomitant with the presence of the DNA of Mycoplasma spp.

It could be concluded that M. bovis and to a lesser extent M. dispar are relatively common in the cattle population of the western part of Iran. Therefore, these pathogens should be taken into consideration whenever respiratory problems are evident in cattle