Keywords : PCR


Detection of Shigella in raw bovine milk by polymerase chain reaction

Noor Soulieman; Aemaan Al-Mariri; Faizah Al-Atrash

Iraqi Journal of Veterinary Sciences, Volume 34, Issue 1, Pages 9-16
DOI: 10.33899/ijvs.2019.125758.1146

Shigella is an intracellular bacterium can infect both human and animal. Its species especially Shigella dysenteriae cause shigellosis worldwide, with 165 million cases of severe bloody diarrhea and mucoid feces. The aim of this study was to find a rapid, sensitive and specific method for screening Shigella in raw bovine contaminated milk. For this goal, 70 samples of milk collected in sterile containers for isolating of Shigella and culturing it on selective media to identify and characterize its morphology, biochemical and molecular characteristics. This study was compared between three different DNA extraction techniques for polymerase chain reaction (direct DNA extraction using a kit, alkaline DNA extraction, and filtrated milk). Our results showed that PCR was able to detect Shigella in 15 out of 15 cases after the milk samples filtered. In other words, the filter technique can be used to detect Shigella in contaminated milk.

Serodiagnosis of Toxocariasis by ELISA test using anti- T. canis IgG antibodies in stray dogs compared to PCR

Noor Jarad; A.K. Abbas; N.N. Aἀiz

Iraqi Journal of Veterinary Sciences, Volume 33, Issue 2, Pages 367-370
DOI: 10.33899/ijvs.2019.163081

Toxocara (T.) canis is a nematode parasite of canines; belong to the Ascarididae family, which accidentally infected humans. Puppies expel the eggs with the feces from the fourth week of the life cycle. This study is the first study in Iraq for detection seroprevalence in stray dogs and extended from January to September 2017. Our study was aimed to investigate the seroprevalence of T. canis infection in stray dogs from different areas in the Al-Diwaniya province, Iraqto detection of specific IgG antibodies to T. canis compared to Conventional PCR technique with the effect of the risk factor. One hundred of the blood sample and one hundred of a faecal sample of same dogs after shooting were studied usingindirect ELISA test and PCR. The result revealed that 71% of the dogs had a seropositive result for this parasite by ELISA test. Dog age is an important factor and affects seroprevalence, were shown that positive rate in adult dogs was more 83.05% than the young dogs 53.65%, while no significant between dogs according to sex. PCR technique showed 58% of dogs were positive forinternal transcribed spacer 1 (ITS1) ribosomal RNA. The sensitivity and specificity of ELISA test was 79 and 40% respectively.

Isolation and identification of Salmonella typhimurium bacteria with detection of type-1 fimbriae coding gene by polymerase chain reaction (PCR) technique

Zeena Saleh; B.M. Al-Muhana; Kh. Hamdan; M.S. Jawad; S.F. Klaif

Iraqi Journal of Veterinary Sciences, Volume 33, Issue 2, Pages 195-199
DOI: 10.33899/ijvs.2019.162961

Two hundred faeces sample were collected from cattle with different age and sex in Al- Diwaniyah Province. The study was conducted in the period between November 2016 and November 2017. Salmonella typhimurium bacteria identified by routine methods such as culturing on selective media, biochemical test and agglutination test using monovalent and multivalent antisera. PCR was can detection type-1 fimbriae gene coding for fimC of Salmonella typhimurium. Results showed that Salmonella isolates were 14.5% in the bovine fecal samples. Also, the serotyping of isolates by using monovalent and polyvalent antisera revealed that all Salmonella isolates in cows were S. typhimurium. The PCR technique was used for detection of type-1 fimbriae coding gene by specific primer for fimC gene. All S. typhimurium isolates in cows appeared to be contained this gene show one distinct band MW.289 bp when electrophoresed on agarose gel. The results of this score indicated that the PCR technique potentate a loud specify in the disclosing of S. typhimurium especially the serotype that encoded to fimC gene type-1 fimbriae isolated from cows in comparison to other routine diagnostic tests.

Microscopic identification, molecular and phylogenetic analysis of Babesia species in buffalo from slaughter house in Al-Najaf city of Iraq

Rashaa Ateaa; Mansour Alkhaled

Iraqi Journal of Veterinary Sciences, Volume 33, Issue 2, Pages 251-258
DOI: 10.33899/ijvs.2019.162882

Babesia is one of hemoprotozoan parasite transmitted by arthropod vectors which responsible for causing of Babesiosis disease in bovine worldwide. The present study was designed for microscopic identification, molecular, and phylogenetic analysis of Babesia species in buffalo from slaughter house in Al-Najaf city of Iraq. The study performed in three months of summer season (August into September 2017) and animals ages and sex were included in this study. The direct microscopic prevalence results were show highest prevalence of haemoprotozoa prevalence at Babesia sp. 45.74%. The prevalence of Babesia sp. related to animal sex, were show in male 43.48% and female was 52%, with non-significant differences. The Prevalence of Babesia sp. related to age were show 12.50%, 92.86% and 30% in young, adult and old age respectively with significant differences (P<0.05). The prevalence of Babesia sp. related to month of study were show. 28.57%, 62.50% and 42.86 in August, September and October respectively and with non-significant differences. Molecular study results were based on PCR and DNA sequencing method by phylogenetic tree analysis (MEGA 6.0) and NCBI-BLAST Homology Sequence Identity to differentiation Babesia species typing. The Babesia species prevalence results were show identified two Babesia species, high prevalence of Babesia bovis (38.30%) were closed related to NCBI-Blast Babesia bovis (HQ264126.1) with homology sequence identity 97-100% and Babesia bigemina 7.45% were closed related to NCBI-Blast Babesia bigemina (KU206291.1) with homology sequence identity 95-99%, then 43 Babesia species includes (B. bovis and B. bigemina) were submitted into NCBI-Genbank and provided accession numbers (MH503811-MH503853). In conclusion, this study concluded that Phylogenetic tree and homology sequences identity was show accurate in differentiation of Babesia species, and these species can be isolated at from local water buffalo from slaughter house in Al-Najaf city, of Iraq.

Detection of Mycoplasma gallisepticum and Mycoplasma synoviae by using of cultural and PCR technique

N.A. Jafar; Bashar Noomi

Iraqi Journal of Veterinary Sciences, Volume 33, Issue 2, Pages 469-473
DOI: 10.33899/ijvs.2019.125484.1016

Laboratory methods are essential for the diagnosis of Mycoplasmal infection. There are three laboratory approaches are essential for the diagnosis of Mycoplasmal infection in chicken including direct methods by culture method and polymerase chain reaction, and indirect methods by detection of Mycoplasmal antibodies by serological tests. This study aimed to detection of Mycoplasma by culture and PCR technique. Two hundred seventy-six samples were collected from infected adult boiler chicken in Salah Al-din province which suffering from respiratory signs and /or joint infection, 202 respiratory and 74 articular samples. According to the results of culture, Mycoplasma isolated in rate of 35.1% (36.6% from respiratory samples and 31.1% from articular samples). The sensitivity of culture was 100%, while the specificity of culture was 97.9% when comparing with PCR results. The current study concluded that the respiratory infection was more than articular infections, and Mycoplasma gallisepticum more distributed than Mycoplasma synoviae among chickens.

First phylogenetic characterization of Pseudocowpox virus from cattle in Al-Qadisiyah province/ Iraq

Salah Mahdi Karim; Khalefa Ali Mansour; Ali Hassan Janabi; Nawras K. M. Al-Nakeeb

Iraqi Journal of Veterinary Sciences, Volume 33, Issue 1, Pages 123-126
DOI: 10.33899/ijvs.2019.125525.1047

This study was initiated for the first time for identification, using sequencing and phylogenetic analyses, of pseudocowpox PCPV that inhabit dairy cows in Al-Qadisiyah province, Iraq. Scab sampling was performed to obtain specimens from udder and teats of 18 affected cows. Initially, a polymerase chain reaction (PCR) method was followed to target a 408-bp piece of the GM_CSF/IL-2 inhibition factor gene (GIF) that belongs to PCPV. Then, the PCR products were sent out to partial sequencing of the GIF gene. The results of the PCR have indicated the presence of the virus in only 3 out of 18 samples. When the sequences were studied using phylogeny, the results have revealed that one of our PCPV strains has a close matching with some of the world strains such as from New Zealand. While two of the current study strains have clustered together with a strain from Finland. The results of our study confirm the presence of the PCPV in dairy cows that induces milker’s nodules.

Phylogenetic tree analysis study of bovine papillomaviruses type 1 based on L1 gene in Al-Qadisiyah governorate, Iraq

Khalefa Ali Mansour; Hassan Hachim Naser; Muthanna Hadi Hussain

Iraqi Journal of Veterinary Sciences, Volume 33, Issue 1, Pages 151-155
DOI: 10.33899/ijvs.2019.125535.1057

Bovine fibropapilloma and papilloma occur in different parts of the skin of animals. Bovine Papillomavirus (BPV) is an oncogenic virus making benign tumor lesion of together mucosal and cutaneous tissue in cattle. In order to confirm the clinical diagnosis; the study planned to make the molecular detection of BPV (DNA) using Polymerase Chain Reaction (PCR) from skin lesions and the phylogenetic analysis. Thirty-eight samples of skin lesions were collected from cattle clinically suspected to be infected with bovine papilloma virus from herds in Al-Qadisiyah Governorate in 2016, the primary clinical diagnosis depended on the morphological appearance and features of the lesion. Deoxyribonucleic Acid (DNA) was extracted from skin lesions; the DNA was examined by PCR technique using specific primer to BPV-1 /L gene-1. Twenty-two samples out of 38 (57,9%), which were collected from different regions in Al-Qadisiyah Governorate, were positive. The sequences of four positive samples of DNA product amplification of (BPV) type-1, L1 gene confirmed the PCR results. These samples had the DNA presented in four accession numbers KY662042-1, KY662043-1, KY662040-1 and KY662041-1. This study proofed that cutaneous bovine papillomatosis related with BPV-1 infection in the cattle herds has affinity to solid skin rather than other epithelial and mucosal tissue.

Sequencing-based phylogenetic-study of Babesia spp detected in tick tissues in Al-Diwaniyah province, Iraq

Marwa Saleem Hajeel; Monyer Abdulameir Abd Alfatlawi

Iraqi Journal of Veterinary Sciences, Volume 33, Issue 1, Pages 9-12
DOI: 10.33899/ijvs.2019.125512.1034

Our study purpose was to investigate the evolution of Babesia spp isolated from tissues of ticks that were found on 150 cows in Al-Diwaniyah province, Iraq. To fulfill the required purpose, sampling of 10 ticks was performed from each infested cow. These obtained ticks were morphologically recognized first, and then they were introduced to Lab investigation that was started with crushing the tick tissues to extract the genomic DNA of the Babesia spp. The DNA was then applied to polymerase chain reaction (PCR) method to recognize the amplification of the region that is related to the 18S rRNA gene. The resulted-amplified products were sequenced for the purpose of confirming and doing the phylogenetic analyses. Here, our study has demonstrated 2 different species according to the results of the sequencing and the phylogenetic analyses of the tested Babesisa species. These 2 species are SP1 and SP2. When the phylogenetic tree was built up, the results showed that SP1 and SP2 are closely related to Babesia bovis (HQ264126.1), an isolate from Texas, USA. Our study indicates interesting and valued data that could be used to study various aspects of the tick, Babesia species, and their control in Al-Diwaniyah City, Iraq.

Molecular study of Anaplasma marginale parasite in carrier cattle in Al-Nasiriyah city

N. R. Al-Kasar; M. M. Flayyih; A. D. Al-Jorany

Iraqi Journal of Veterinary Sciences, Volume 32, Issue 2, Pages 299-301
DOI: 10.33899/ijvs.2019.153867

To detect Anaplasma marginale among carrier cattle by using polymerase chain reaction (PCR) technique, 64 blood samples, fromhealthy cows in abattoir of Al-Nasiriyah city were collected from June till August, 2017 in this study. By targeting MAR1bB2 gene with the molecular weight of approximately 265 bp, Anaplasma marginale were detected in18 samples (28.125%). One of these positive sample was recoded in National Center for Biotechnology Information, NCBI; Gene Bank.

Use molecular techniques as an alternative tool for diagnosis and characterization of Theileria equi

M.A. El-Seify; N.M. Helmy; N.M. Elhawary; Sh.S. Sorour; A.M. Soliman

Iraqi Journal of Veterinary Sciences, Volume 32, Issue 1, Pages 5-11
DOI: 10.33899/ijvs.2018.153787

The purpose of this study was to determine the prevalence of clinical, subclinical and chronic infection with the equine parasite T. equi in some Egyptian localities (Cairo and Giza governorates). A panel of 396 equine blood samples representing 141 horses, 250 donkeys and 5 mules was collected from equines during the period from April 2015 to March 2016 using microscopic examination and conventional PCR. Microscopically a twenty two (5.56%) of 396 were positive for T. equi merozoites that appeared as small rounded, pyriform shaped and maltase cross shaped merozoites. Among 8/141(5.67%) horses and 14/250 (5.60%) donkeys were found to have positive for T.equi. A one hundred blood samples (45 horses, 50 donkeys and 5 mules) selected randomly were also examined by PCR. The results of PCR showed 30/100(11/45 (24.4%) horses, 18/50 (36%) donkeys and 1/5 (20%) mule) were positive for T.equi. When the sequenced PCR amplicons (n=3) were aligned to the reference nucleotide sequences of T. equi accessed in Genbank, the horse isolate showed insertion of Thymine (T) base at position 23 and substitution of Thymine (T) base with Cytosine (C) base at position 91, while the donkey and mule isolates have no alterations when compared to the reference sequences. The phylogenetic analysis showed that the sequenced PCR isolates belonged to T. equi. The obtained sequences were deposited in the GeneBank database under accession numbers MF192854, MF192855 and MF192856.

Polymorphism of growth hormone gene in the artificial insemination result of Madura cattle with Limousin semen as a reference for genetic selection

B. Utomo; E. Safitri

Iraqi Journal of Veterinary Sciences, Volume 32, Issue 1, Pages 113-118
DOI: 10.33899/ijvs.2018.153832

Research on genetic polymorphism of growth hormone (GH) and receptor growth hormone (rGH) has not been done in crossbred of Limousin cattle, so it is interesting to be examined. Blood samples were taken from 14 Madura calves were artificially inseminated with Limousin cement. DNA amplification is done by using Polymerase Chain Reaction (PCR) method, Restriction Fragment Length Polymorphism (RFLP) method to determine the genotype. DNA sequencing was done to determine nucleotide sequences of GH unit genes. The results showed that identification of GH and rGH gene polymorphisms was done by breaking DNA fragments from 432 and 298 bp in Madura and Limousin cattle (Madrasin) ie, L and V alleles have a frequency of 0.67 and 0.33 for the GH gene, respectively. This proves that the crossed-breeding of Madrasin have V allele that is not owned by the Madura cattle. While in the rGH gene, the A allele is 0.92 and the G allele is 0.08, with the frequency of the A allele larger than the G allele. This research concluded: that GH and rGH undergo changes on polymorphisms in Madrasin cattle can be used as a basis for selection.

Prevalence and molecular studies on Echinococcus equinus isolated from necropsied donkeys

A.Y. Desouky; N.M. Helmy; Sh.S. Sorour; M.M. Amer

Iraqi Journal of Veterinary Sciences, Volume 31, Issue 2, Pages 101-106
DOI: 10.33899/ijvs.2017.145605

In the present study, forty donkeys of different ages and sexes at Giza Zoo, Egypt were investigated between October 2015 and September 2016 for the presence of hydatidosis disease. Hydatid cysts were detected in the livers of 10% of the examined donkeys and these cysts had a fertility rate 100%. Female donkeys were infected with cysts more than males and all infected donkeys were old aged with no cases of infection were detected in young or adult donkeys. Using molecular tools, the DNA extracted from cysts that had been isolated was subjected to PCR amplification, using synthesized oligonucleotide primers, and these were constructed to target the 299 bp within the (ND2) gene, which is considered to be specific for the Echinococcus equinus genotype. The sequenced PCR products showed homology to E.equinus (G4 or horse strain genotype). These results can be used in future to pursue the epidemiological status of the causative strain of hydatidosis in equines at the study area.

Phylogenetic study of Theileria lestoquardi based on 18SrRNA gene Isolated from sheep in the middle region of Iraq

M.J.A. Alkhaled; N.N. A'aiz; H.H. Naser

Iraqi Journal of Veterinary Sciences, Volume 30, Issue 2, Pages 27-32
DOI: 10.33899/ijvs.2016.121380

Theileriosis is parasitic infection causes by obligate intracellular protozoa of the genus Theileria. T. lestoquardi is the most virulent species in sheep and goats which causes a severe disease with a high morbidity and mortality rate. In this study the phylogenetic relationships between two local isolate of T. lestoquardi and nine T. lestoquardi global isolates as well as Babesia ovis out-group isolate were analyzed using the 18S rRNA gene sequence. The multiple sequence alignment analysis and neighbor joining phylogenetic tree analysis were performed by using ClustalW multiple sequence alignment online based analysis of 1098bp 18S rRNA gene was amplified by polymerase chain reaction. Phylogenetic analysis results of these gene sequences revealed that T. lestoquardi local isolates were closely related to T. lestoquardi Iran isolate (JQ917458.1) and two Iraq Kurdistan isolates (KC778786.1 and KC778785.1) more than other countries. This study represents the first report on the use of molecular phylogeny to classify T. lestoquardi obtained in Middle Region of Iraq.

Using species-specific PCR technique to detect Toxoplasma gondii in broiler chickens

R.A. Al-Sanjary; T.H. Hussein

Iraqi Journal of Veterinary Sciences, Volume 26, Issue 2, Pages 53-56
DOI: 10.33899/ijvs.2012.67452

Two groups of broiler chickens were used in this study. One was reared under typical conditions at the animal house of Veterinary Medicine College/Mosul University- Iraq, while the other group was reared under common commercial farm conditions. Fifty and 80 birds from the two respective groups were sacrificed at 49 days of age for detecting Toxoplasma gondii by using Species-specific PCR technique. Results of Latex agglutination test indicated, principally, that 29.3% and 49.2% of the serum samples were positive for the birds of both groups, respectively. Titer figures ranged between 1:20 to 1:320 where the highest value was 1:160 (39.3%) and the lowest was 1:20 (5.8%). Confirmation of 38 and 64 serum samples, using Latex agglutination test was performed by PCR technique, from the two respective groups of chickens. Of those, 8 samples from the college birds and 35 from the commercial farm birds were confirmed positive by giving band of 133 bp, according to specific primers designated on gene B1.Based on these results, pursuing the PCR technique is considered, so far, a most sensitive method for Toxoplasma gondii detection. Also, positive PCR results are counted on as an early marker for reactivation and useful means in monitoring therapies.