BACKGROUND: Causing site direct mutation can be one of the efficient methods to evaluate the characteristics and properties of various genes. Brucellosis is the most common zoonotic infectious disease that would cause great economic losses. Thus, recognition of pathogenic and immunogenic factors in the genus Brucella can lead to control this health problem. Objectives: Considering the importance of site direct mutation in identification of genome structure and numerous ways to achieve this goal, Overlap Extension PCR is introduced as an improved technique for the removal and replacement of the gene target. Methods: For this study, with two-step PCR using specific primers, upstream and downstream fragments from target gene and antibiotic resistance cassette from plasmid pET28a (+), were reproduced and were connected to each other. The resulting fragment was cloned in specific position of pBluescriptIISK(-) plasmid by the restriction enzymes. Then, the construction was transferred into the genome of Brucella abortus by electroporation method. Results: Fusion PCR product was obtained without any change in the nucleotide sequence and then it was cloned into pBluescriptIISK (-) plasmid, finally the construction was replaced and the target gene was deleted. Conclusions: The results of this study show that the Overlap Extension PCR is an optimized and modified technique to create mutations in the bacterial genome structure and can easily be used in the family Brucella.

The lack of proofreading activity of the viral polymerase and the segmented nature of the influenza A virus (IAV) genome are responsible for the genetic diversity of IAVs and for their ability to adapt to a new host. We tried to adapt avian IAV (avIAV) to the pig by serial passages in vivo and assessed the occurrence of point mutations and their influence on viral fitness in the pig’s body.

Porcine circovirus type 3 (PCV3) is a newly discovered porcine circovirus. The molecular characteristics and genetic evolution of PCV3 in Xinjiang province, China still being unclear, the aim of the study was their elucidation. A total of 393 clinical samples were collected from pigs on commercial farms in nine different regions of Xinjiang and phylogenetic analysis based on full-length Cap genes was performed. The prevalence at farm level was 100%, while in all the tested samples it was 22.39%. Nine PCV3 strains were detected in Xinjiang province and they shared 98.9–99.3% nucleotide and 97.5–100.0% Cap gene amino acid sequence identities with other epidemic strains from China and abroad. Compared with other epidemic strains of PCV3, there were 26 base mutation sites in the Cap gene in the nine Xinjiang strains, resulting in the mutation of amino acids at positions 20, 24, 75, 77, 108, 111 and 206. Phylogenetic analysis showed that these strains can be divided into two different genetic groups, to the first of which five strains affiliated and divided between subgroups 1.1 and 1.2, and to the second of which the other four strains affiliated and similarly divided between subgroups 2.1 and 2.2. PCV3 circulates widely among commercial pig farms in Xinjiang province, China, and displays obvious genetic diversity. The results provide epidemiological information useful for the prevention and control of PCV3 infection in the pig industry.

Introduction: Mycoplasma bovis is one of the main pathogens involved in cattle pneumonia. Other mycoplasmas have also been directly implicated in respiratory diseases in cattle. The prevalence of different Mycoplasma spp. in cattle affected by respiratory diseases and molecular characteristics of M. bovis field strains were evaluated. Material and Methods: In total, 713 nasal swabs from 73 cattle herds were tested. The uvrC gene fragment was amplified by PCR and PCR products were sequenced. PCR/DGGE and RAPD were performed. Results: It was found that 39 (5.5%) samples were positive for M. bovis in the PCR and six field strains had point nucleotide mutations. Additionally, the phylogenetic analysis of 20 M. bovis field strains tested with RAPD showed two distinct groups of M. bovis strains sharing only 3.8% similarity. PCR/DGGE analysis demonstrated the presence of bacteria belonging to the Mollicutes class in 79.1% of DNA isolates. The isolates were identified as: Mycoplasma bovirhinis, M. dispar, M. bovis, M. canis, M. arginini, M. canadense, M. bovoculi, M. alkalescens, and Ureaplasma diversum. Conclusion: Different Mycoplasma spp. strains play a crucial role in inducing respiratory diseases in cattle.

The lack of proofreading activity of the viral polymerase and the segmented nature of the influenza A virus (IAV) genome are responsible for the genetic diversity of IAVs and for their ability to adapt to a new host. We tried to adapt avian IAV (avIAV) to the pig by serial passages in vivo and assessed the occurrence of point mutations and their influence on viral fitness in the pig’s body. A total of 25 in vivo avIAV passages of the A/duck/Bavaria/77 strain were performed by inoculation of 50 piglets, and after predetermined numbers of passages 20 uninoculated piglets were exposed to the virus through contact with inoculated animals. Clinical signs of swine influenza were assessed daily. Nasal swabs and lung tissue were used to detect IAV RNA by real-time RT-PCR and isolates from selected passages were sequenced. Apart from a rise in rectal temperature and a sporadic cough, no typical clinical signs were observed in infected pigs. The original strain required 20 passages to improve its replication ability noticeably. A total of 29 amino-acid substitutions were identified. Eighteen of them were detected in the first sequenced isolate, of which 16 were also in all other analysed strains. Additional mutations were detected with more passages. One substitution, threonine (T) 135 to serine (S) in neuraminidase (NA), was only detected in an IAV isolate from a contact-exposed piglet. Passaging 25 times allowed us to obtain a partially swine-adapted IAV. The improvement in isolate replication ability was most likely related to S654 to glycine (G) substitution in the basic protein (PB) 1 as well as to aspartic acid (D) 701 to asparagine (N) and arginine (R) 477 to G in PB2, glutamic acid (E) 204 to D and G239E in haemagglutinin and T135S in NA.

Cysticercosis caused by the larval stage of Taenia hydatigena is economically the most important endemic parasitic disease in Iraq. Few data are available relating to the genetic divergence of this helminth. This study aimed to molecularly characterise Cysticercus tenuicollis isolates from sheep in Sulaymaniyah province, Iraq. DNA extraction and amplification of specimens of C. tenuicollis from 46 sheep were conducted by PCR for the mitochondrial 12S rRNA gene. The 19 amplicons were subjected to purification and partial sequencing. Five 12S rRNA nucleotide sequence haplotypes were found. The pairwise nucleotide difference between haplotypes of 12S rRNA gene ranged from 0.2% to 0.7%. Four out of the five haplotypes of C. tenuicollis contained one to two base mutations and were discovered in Iraq for the first time, and this may be a unique mutation globally which has not been recorded previously. Three newly recorded haplotypes contained only one single mutation, and the other one contained two mutations. Phylogenetic analysis showed that all isolated strains were closely related to Iranian sheep isolates. Four new strains of T. hydatigena were discovered for the first time in the study area.

Human coronaviruses are known respiratory pathogens associated with a range of respiratory illnesses, and there are considerable morbidity and hospitalisation amongst immune-compromised individuals of all age groups. The emergence of a highly pathogenic human coronavirus in China in 2019 has confirmed the long-held opinion that these viruses are important emerging and re-emerging pathogens. In this review article, we trace the discovery and emergence of coronaviruses (CoVs) over time since they were first reported. The review article will enrich our understanding on the host range, diversity and evolution, transmission of human CoVs and the threat posed by these viruses circulating in animal populations but overtime have spilled over to humans because of the increased proximity between humans and animals.

Porcine reproductive and respiratory syndrome (PRRS), which is caused by the PRRS virus (PRRSV), has resulted in large economic losses for the swine industry. The virus has shown remarkable genetic diversity since its discovery. In our study, we investigated mutation types in the evolution of PRRSV for both in vivo and in vitro passaging of the virus. Sequence alignment analysis demonstrated that the most common hypermutations expressed were A→G/U→C and G→A/C→U. The data provide a new theoretical basis for PRRSV evolution.

OBJECTIVE To characterize clinical findings for polysaccharide storage myopathy (PSSM) in warmblood horses with type 1 PSSM (PSSM1; caused by mutation of the glycogen synthase 1 gene) and type 2 PSSM (PSSM2; unknown etiology). SAMPLE Database with 3,615 clinical muscle biopsy submissions. PROCEDURES Reported clinical signs and serum creatine kinase (CK) and aspartate aminotransferase (AST) activities were retrospectively analyzed for horses with PSSM1 (16 warmblood and 430 nonwarmblood), horses with PSSM2 (188 warmblood and 646 nonwarmblood), and warmblood horses without PSSM (278). Lameness examinations were reviewed for 9 warmblood horses with PSSM2. Muscle glycogen concentrations were evaluated for horses with PSSM1 (14 warmblood and 6 nonwarmblood), warmblood horses with PSSM2 (13), and horses without PSSM (10 warmblood and 6 nonwarmblood). RESULTS Rhabdomyolysis was more common for horses with PSSM1 (12/16 [75%] warmblood and 223/303 [74%] nonwarmblood) and nonwarmblood horses with PSSM2 (221/436 [51%]) than for warmblood horses with PSSM2 (39/147 [27%]). Gait abnormality was more common in warmblood horses with PSSM2 (97/147 [66%]) than in warmblood horses with PSSM1 (1/16 [7%]), nonwarmblood horses with PSSM2 (176/436 [40%]), and warmblood horses without PSSM (106/200 [53%]). Activities of CK and AST were similar in warmblood horses with and without PSSM2. Muscle glycogen concentrations in warmblood and nonwarmblood horses with PSSM1 were significantly higher than concentrations in warmblood horses with PSSM2. CONCLUSIONS AND CLINICIAL RELEVANCE Rhabdomyolysis and elevated muscle glycogen concentration were detected in horses with PSSM1 regardless of breed. Most warmblood horses with PSSM2 had stiffness and gait abnormalities with CK and AST activities and muscle glycogen concentrations within reference limits.

Myocardial dysfunction occurs in cats with hypertrophic cardiomyopathy (HCM), but little is known about the early stages of the disease. Strain imaging echocardiography is a method that enables the quantitative assessment of myocardial function and deformity, allowing the characterization of systolic dysfunction. The objective of this study was to assess systolic function using strain imaging echocardiography in Maine coon cats genetically tested for the A31P mutation in the MYBPC3 gene, with and without ventricular hypertrophy. For this purpose, 57 Maine coon cats of both genders, with an unknown status regarding the mutation at inclusion, were included prospectively and evaluated by conventional and strain imaging echocardiography. Comparisons were made among cats without hypertrophy (n = 45), suspect cats (n = 7), and cats with hypertrophic cardiomyopathy (n = 5), and also between the heterozygous for the mutation group (n = 26) and the negative for the mutation group (n = 28). Finally, in the group of phenotypically normal cats, heterozygous cats carrying the mutation were compared to cats without the mutation. Strain values were compared among the groups (blinded prospective study). While echocardiography demonstrated normal contractility, strain values (middle of the septum) were lower in HCM cats. Strain values (base of anterior wall of the left ventricle) were lower in heterozygous than in negative cats, even before hypertrophy. Negative correlation was observed between some values of myocardial strain and thickness. While strain imaging echocardiography was able to detect systolic abnormalities, despite apparent normality on conventional echocardiography, it was not able to identify cats that carry the A31P mutation in the MYBPC3 gene. Strain imaging echocardiography could be a useful tool, however, for detecting systolic alterations in HCM cats with an apparently normal systolic function or for detecting alterations in normal carriers of the MYBPC3 gene mutation.