Biomarker-Based Evaluation and Molecular Identification with Phylogenetic Analysis of Echinococcus granulosus from Liver Samples of Sheep and Goats in Al-Qadisiyah Province, Iraq

Trichostrongyles are common causes of parasitic gastroenteritis in sheep and goats worldwide. Accurate identification of these nematodes to the genus and/or species level is important for therapy selection and control strategies. In the present study, molecular and egg-lectin binding approaches were employed to identify the most economically important trichostrongyles circulating in sheep and goat herds from six districts in Dakahlia governorate, Egypt. Fecal samples from 653 and 205 goats reared within 17 herds were collected and tested for the trichostrongyle eggs using the modified Wisconsin sucrose flotation method. For identification of the trichostrongyle(s) present, eggs from 75 (63 sheep and 12 goats) samples which had high egg count (EPG) and pooled eggs (n = 19 pools, 15 sheep and 4 goats) from samples with moderate or low EPGs were examined. Molecular examination was conducted amplifying the ITS2 region of the rDNA for six different trichostrongyles in individual PCR reactions. For egg-lectin bindings, 4 fluorescently-labeled specific lectins were used; peanut agglutinin (PNA) for Haemonchus contortus, Aleuria aurantia agglutinin (AAL) for Trichostrongylus species, Lens culinaris agglutinin (LCA) for Teladorsagia circumcnicta and Lotus tetragonolobus lectin (LTL) for Cooperia species. Fourteen (82.3%) herds were found infected, of which trichostrongyle eggs were detected in fecal samples of 26.5% (173/653) of sheep and 10.2% (21/205) of goats. Results of the PCR and lectin bindings were compatible and 4 trichostrongyles were detected: H. contortus, T. circumcincta, Trichostrongylus axei and Trichostrongylus colubriformis. Haemonchus contortus eggs were found in all the infected herds, and as the single species in 21 and 5 of sheep and goat samples, respectively. Lectin stained smears demonstrated the dominance of H. contortus eggs over eggs of the other detected trichostrongyles. Eleven herds were found infected with T. axei as the second most prevalent trichostrongyle; however, few AAL-stained eggs were noticed in the positive samples. Mixed infections were frequently detected as H. contortus-T. axei combination. Infections with T. circumcincta were noted in sheep samples from two herds, but not in any sample from the goats. No Ostertagia leptospicularis, Cooperia curticei or Nematodirus species were noted among the tested samples. This is the first molecular and lectin binding survey to determine the species composition of trichostrongyles infecting sheep and goats from Egypt. Haemonchus contortus plays the principal role in small ruminant trichostrongylosis in Egypt. Egg-lectin staining shows promise for future for its application in routine diagnosis as a rapid and simple technique. Findings of the earlier reports from Egypt are tabulated and reviewed.

Molecular characterization of Cysticercus tenuicollis of slaughtered livestock in Upper Egypt governorates

The present study was conducted during the period from September 2015 until February 2016. 100 fecal samples were collected from 60 sheep and 40 goats for diagnosis of Cryptosporidium parasite from diverse areas in Al-Qadisiyah province. The study amid to know the genetic characters of Cryptosporidium spp. parasite by using a molecular technique such as the nested polymerase chain reaction and DNA sequencing analyzed by phylogenetic tree to identify the parasite species. This study was done on the sheep and goat at first time in the middle region of Iraq and the identified species were recorded in NCBI-Genbank database. In sheep, the results of positive infected samples was (40%) while, in the goats were (32.5%), the DNA sequencing and phylogenetic analysis method based on small ribosomal RNA gene (18s rRNA) for Cryptosporidium species typing. The results were conducted by Neighbor-Joining phylogenetic tree analysis method and the 18s rRNA gene sequences were confirmed by using NCBI-BLAST data analysis in order to compare with NCBI submitted selected references isolates of (18s ribosomal RNA) gene in Cryptosporidium spp. parasites. Our finding in present study appeared to follow spp. (C. parvum, C. hominis, C. andersoni, C. ubiquitum, C. xiaio and C. suis). These identified species which primary affected sheep and goats as mentioned in previous studies when compared with newly Iraq isolates strains.

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.

Clostridium perfringens (C. perfringens) belongs to the family of Clostridiaceae and produces a wide range of toxins (four major and a variety of minor toxins). Some toxins associated with virulence have been shown to participate in the pathogenesis of enteric diseases in sheep, goats, and other animals. The aim of this study was to determine the presence of minor virulence genes and their genetic diversity in the various toxinotypes of C. perfringens isolates. About 84 isolates collected from sheep and goat flocks were examined and sequenced for the presence of minor virulence genes. Results showed that PFO and cpb2 were found in 79 out of 84 (94%), cpe was identified in 29 out of 84 (35%) and the presence of tpeL was confirmed in 28 out of 84 (33%) isolates, while none of the isolates were identified as carrying the netB gene. This study shows that the prevalence of genes varied among various types of C. perfringens isolates and also sheep and goat samples, furthermore these findings change the toxinotypes of isolates based on a modified scheme of toxinotype, which incorporated CPE and NetB toxins. The results of this study showed that the dominant minor virulence genes were PFO and cpb2 and the occurrence of cpe and tpeL genes was also diverse. DNA sequencing revealed approximately a sequence similarity of 97-100% with the GenBank database and 3 motation was found in sequence analysis.

Seroepidemiological study of Toxoplama gondii in small ruminants (sheep and goat) in different provinces of Mongolia

Serological evidence for the presence of influenza D virus in small ruminants

BackgroundTo develop an effective malaria vector intervention method in forested international border regions within the Greater Mekong Subregion (GMS), more in-depth studies should be conducted on local Anopheles species composition and bionomic features. There is a paucity of comprehensive surveys of biodiversity integrating morphological and molecular species identification conducted within the border of Laos and Cambodia.MethodsA total of 2394 adult mosquitoes were trapped in the Cambodia–Laos border region. We first performed morphological identification of Anopheles mosquitoes and subsequently performed molecular identification using 412 recombinant DNA–internal transcribed spacer 2 (rDNA-ITS2) and 391 mitochondrial DNA–cytochrome c oxidase subunit 2 (mtDNA-COII) sequences. The molecular and morphological identification results were compared, and phylogenetic analysis of rDNA-ITS2 and mtDNA-COII was conducted for the sequence divergence among species.ResultsThirteen distinct species of Anopheles were molecularly identified in a 26,415 km2 border region in Siem Pang (Cambodia) and Pathoomphone (Laos). According to the comparisons of morphological and molecular identity, the interpretation of local species composition for dominant species in the Cambodia–Laos border (An. dirus, An. maculatus, An. philippinensis, An. kochi and An. sinensis) achieved the highest accuracy of morphological identification, from 98.37 to 100%. In contrast, the other species which were molecularly identified were less frequently identified correctly (0–58.3%) by morphological methods. The average rDNA-ITS2 and mtDNA-COII interspecific divergence was respectively 318 times and 15 times higher than their average intraspecific divergence. The barcoding gap ranged from 0.042 to 0.193 for rDNA-ITS2, and from 0.033 to 0.047 for mtDNA-COII.ConclusionsThe Cambodia–Laos border hosts a high diversity of Anopheles species. The morphological identification of Anopheles species provides higher accuracy for dominant species than for other species. Molecular methods combined with morphological analysis to determine species composition, population dynamics and bionomic characteristics can facilitate a better understanding of the factors driving malaria transmission and the effects of interventions, and can aid in achieving the goal of eliminating malaria.Graphical

More than 30% of fruits of Chinese Quince (Chaenomeles speciosa) and peach (Prunus persica) showed circular, water-soaked and brown spots in July 2022 in Kunming, Yunnan, China. The center of these spots was covered by a large number of earthy brown and oblate sporogeneous mycelium containing conidiophore and conidia, which were one-celled, limoniform, hyaline (13.73 to 22.77 x 8.17 to 12.84 µm, n=50). By September 2022, almost 100% of fruits showed symptoms. Later, most of them fell or a few stiff, black and mummified fruits were left on the trees. Fungal isolates were isolated by single-spore technique on Potato Dextrose agar (PDA) from the diseased fruits, and incubated at room temperature (20-28 °C) in darkness for 14 days. The colony was gray, smooth at margins, 7.6-8.0 cm in diameter. To fullfill Koch's postulates, mycelial plugs of one representative isolate YHD611 from Chinese Quince and another YHD610 from peach were used to inoculate three wounded and three non-wounded surface-disinfected fruits of both hosts at room temperature (19-27 °C), respectively. Three wounded and three non-wounded fruits inoculated with sterile PDA plugs served as the control. The wounded peaches appeared water-soaked and had brown lesions after three days of inoculation, then completely decayed after nine days, while non-wounded fruits showed symptoms after five days. The wounded fruits of Chinese Quince developed similar symptoms after eight days of inoculation, and completely decayed after 13 days, while non-wounded fruits showed obvious symptoms after 15 days. In a subsequent study, isolate YHD611 was inoculated to peach while isolate YHD610 was inoculated to Chinese Quince to understand host specificity of the isolates. The results showed that when peaches were infected with YHD611, symptoms were observed on wounded fruits after three days while on non-wounded fruits after five days. When Chinese Quince was infected with YHD610, symptoms were observed on wounded fruits after 14 days while on non-wounded fruits after 21 days. Fungal isolates from symptomatic fruits were identical to the original isolates. There were no symptoms on the control fruits of both hosts. Molecular identification was confirmed based on the sequences of internal transcribed spacer (ITS, primers ITS1 and ITS4) and β-tubulin (TUB2, primers Bt2a and Bt2b) genes (Niu et al. 2016). BLASTn analysis of the ITS (OQ15519and OQ155196) and TUB2 (OQ185202 and OQ185201) of YHD611 and YHD610 revealed a 100% sequence identity, respectively, to Monilia yunnanensis AH7-2 (KT735924.1 for ITS, KT736008.1 for TUB2). In the phylogenetic analyses based on ITS and TUB2 sequence data, the isolates YHD611 and YHD610 belonged to the M. yunnanensis clade. Based on morphological and molecular identification, both isolates were identified as M. yunnanensis, which was reported as the pathogen causing brown rot of plum, peach, apple and pear in Yunnan, China (Hu et al. 2011; Yin et al. 2015). To our knowledge, this is the first report of M. yunnanensis causing brown rot on the fruits of Chinese Quince in Yunnan, China. This study also reports that M. yunnanensis from Chinese Quince can infect peach, and the pathogen from peach can infect Chinese Quince. These findings suggest that M. yunnanensis can transfer from one host to another and causing serious economic losses in multiple fruit crops in Yunnan, China.

Molecular detection of the B1 gene of Toxoplasma gondii in blood samples of female sheep and goats in Tebessa, northeastern Algeria

This study aimed to identify ovine herpesvirus 2 (OHV-2) infections in sheep and goats in Al-Qadisiyah Province of Iraq, using molecular and phylogenetic methods. Nasal discharge swabs were collected from 60 sheep and 60 goats from 3 different animal sale bars. The samples were subjected to semi-nested-polymerase chain reaction (PCR), sequencing, and phylogenetic tests involving OHV-2 tegument protein gene (OHV-2T). The results of the semi-nested PCR showed the presence of OHV-2 in all 60 (100%) sheep and 52 (86.6%) goats. The samples from both sheep and goats were sent for partial-gene-based sequencing to confirm the PCR results. Phylogenetic analysis was conducted and 6 PCR amplicons (10%) of positive samples from each goat and sheep were submitted for sequencing. The sequence results were reassembled and deposited in the NCBI-GenBank database under the accession numbers of MF004402.1 for sheep and MG875327.1 and MG875328.1 for goats. Multiple alignments of sequences showed close identities with some global isolates of this virus. This study not only reports new sequences from the local OHV-2 isolates that have been deposited in the NCBI GenBank, but also provides important data about the presence and shedding of OHV-2 in the nasal discharge of healthy sheep and goats, and suggests OHV-2 as the major cause of malignant catarrhal fever in cattle.

Background: Listeria monocytogenes, a zoonotic pathogen affecting humans and animals, exhibits a global distribution, including Iraq. This study focused on the rapid identification and phylogenetic analysis of L. monocytogenes in freshly collected ewe milk samples from various farms in Al-Qadisiyah province, Iraq.Methods: The study was conducted with care and precision, involving 150 milk samples. These samples were subjected to traditional bacterial isolation and identification using the enrichment culture method and biochemical tests, with the PCR technique confirming the results. The antibacterial activity of MgONPs was then assessed using the disc diffusion method, ensuring a comprehensive and reliable approach to the study.Result: The results show that 150 ewe milk samples underwent real-time PCR (RT-PCR) targeting the ssrA gene, followed by partial 16S rRNA gene sequencing (PSGS) of purified conventional PCR products. Furthermore, the study entails the biosynthesis of magnesium oxide nanoparticles using Myrtus communis leaf extract, followed by a comprehensive characterization utilizing UV-spectra, FTIR, SEM, and TEM techniques. The Agar well diffusion method assessed the antibacterial efficacy of these Biosynthesized nanoparticles against L. monocytogenes. The RT-PCR results revealed the presence of L. monocytogenes in 36 out of 150 samples (24%). Subsequent PCR analysis confirmed the presence of the pathogen in 30 out of these 36 positive samples (83.33%). Sequencing of two purified PCR products demonstrated 100% nucleotide identity with global isolates from Iraq and Turkey. Furthermore, the study demonstrated that L. monocytogenes exhibited substantial sensitivity (24.66 ± 0.3) to the biosynthesized magnesium oxide nanoparticles. These findings underscore the speed and precision of the RT-PCR method for detecting L. monocytogenes in fresh ewe milk samples.Conclusion: This comprehensive investigation enhances our understanding of L. monocytogenes prevalence in ewe milk and highlights the potential of Myrtus communis -derived nanoparticles for combating this pathogen.Keywords: Antibacterial nanoparticles; Listeria monocytogenes; Magnesium oxide; Myrtus communis sheep milk; ssrA gene

Anaplasma spp. are widely spread rickettsial bacteria transmitted by ticks and placing high impacts on veterinary and public health. A limited number of studies have been carried out on Anaplasmosis in the central part of Iraq. This study was conducted to determine the presence of Anaplasma spp. in cattle in Al-Qadisiyah province, Iraq. A total of 400 blood specimens were collected from cattle suffering from heavy tick infestation. Cattle were blood-sampled from four hyper-endemic areas with ticks. Blood samples were screened using microscopic and polymerase chain reaction (PCR) methods. Diff-quick stained blood smears revealed Anaplasma-like inclusion bodies in 254 (63.5%) samples. According to the 16S rRNA-gene-based PCR analysis, Anaplasma spp. was detected in 124 of the 400 (31%) samples, divided as 96/254 (37.8%) among the microscopical positive samples and 28/146 (19.17%) among the microscopical negative samples. Phylogenetic analysis based on the partial 16S rRNA gene sequencing of ten-PCR positive samples were 99–97% identical to sequences deposited in the GenBank, revealing presence of A. phagocytophilum, A. marginale and unnamed Anaplasma spp. in 40%, 20%, and 40% samples, respectively. Relationships among Anaplasma spp. infections and cattle breed, age, and sex were analyzed. Calves less than one year old showed significantly higher rates (p<0.005) than those from other age groups, whereas sex and breed demonstrated no significant differences (p˃0.001). This study shows that a variety of Anaplasma spp., were endemic in central part of Iraq and is still a hidden problem in cattle in the hyperendemic areas of tick, which requires serious control strategies.

The aim of this study which conducted in Al-Diwaniyah province during the period from October (2021) to April (2022) was to study histopathological examination of intestine and molecular identification with phylogenetic analysis of Eimeria. A total of 25 tissue samples (small intestine and large intestine) of goat were collected from slaughter houses in Al-Diwaniyah province. Histopathological study of the intestine revealed immature oocyst of the parasite in the colon, and the reproductive stages of the parasite were observed, represented by the presence of female (macrogametes) and male (microgametes), in addition to observing some histopathological changes represented by the presence of infiltration of lymphocyte and eosinophil of villi in goat. Genomic DNA was extracted from 100 goat´s fecal samples and18S rRNA gene of Eimeria was amplified by polymerase chain reaction. PCR technique showed that, out of 100 goat´s fecal samples 87(87%) were positive for 18S rRNA gene of Eimeria. Fifteen PCR positive product of local Eimeria isolates were sent to Bioneer company in Korea for detected the DNA sequence and compared it with that of other Eimeria published in GenBank.

Phylogenetic tree analysis study of Lumpy skin disease virus based envelope protein P32 gene in Al-Qadisiyah province, Iraq