Background: The dairy sector faces formidable obstacles in the early detection of the bacteria causing subclinical mastitis and in the proactive treatment of those cases. The objective of the study was to access multiplex PCR assays (MPCR) and conventional methods for the concurrent identification of significant bacteria that cause sub-clinical mastitis and to compare these methods. 200 samples of pooled milk from cows and buffaloes in Rajasthan were gathered between 2020 and 2021.&nbsp; 
 
Methods: The primary bacterial pathogens in milk samples were identified using conventional methods such multiplex PCR, biochemical testing and culture.&nbsp; 
 
Result: From 200 combined samples of cow and buffalo milk, the traditional approach was able to extract 97 different strains. Staphylococcus aureus 54 (27%), Streptococcus spp. 30 (15%) and E. coli 13 (6.5%) were shown to be prevalent as single or mixed infections, respectively. Staphylococcus was the main pathogen that was discovered. concurrently, S. aureus, Streptococcus and E. coli. Direct detection of Staphylococcus 65 (32.5%), Streptococcus 37 (18.5%), E. coli and 16 (8%) by multiplex PCR was found in milk samples. The analysis revealed that because multiplex PCR assays have higher specificity and sensitivity than conventional procedures, they are more reliable. The multiplex PCR method employed in the current study was a simple and quick technique to identify the major pathogens and it has the potential to be a very helpful tool for determining the pathogens that cause environmental mastitis and evaluating the health of the herd.

Molecular Detection of Some Most Important Pathogenic Bacteria by Multiplex PCR for Diagnosis of Subclinical Mastitis in Bovine

<div>Background: Antimicrobial resistance (AMR) is a rising concern in the global aquaculture sector due to the rampant prophylactic use of antibiotics. This study is aimed to determine the AMR pattern in freshwater ornamental fishes.&nbsp; 
 
Methods: Fish pathogens were isolated and identified from infected Guppy and Molly collected from ornamental fish farms. Antibiotic susceptibility testing (ABST) using disc diffusion with 36 antibiotics was performed following the disc diffusion method. The resistance gene cassettes such as Class 1 and Class 2 integron were also detected from resolved isolates.&nbsp; 
 
Result: Fish pathogens were isolated and identified as Aeromonas hydrophila, A. dhakensis, A. veronii, A. sobria, Bacillus subtilis, Comamonas testosteroni, Edwardsiella tarda, Enterobacter cloacae, Enterococcus faecalis, Kurthia gibsonii and Klebsiella aerogenes. Shannon Weiner diversity index of resolved isolates was found to be 1.366 and 2.101 for Guppy and Molly, respectively. ABST results showed an elevated resistance pattern for A. veronii, E. faecalis (Guppy), K. aerogenes, B. subtilis, E. faecalis, C. testosteroni (Molly) with higher multiple antibiotic resistance indexes (&gt;0.33). Meanwhile, all the recovered isolates were susceptible to sulphafurazole, enrofloxacin, norfloxacin and ciprofloxacin. Detection of class 1 integron in genomic and plasmid DNA reminds the rapid spread of the AMR gene through horizontal gene transfer.</div>

Prevalence, Antimicrobial Resistance and Resistance Gene Cassettes Detection in Bacterial Pathogens Isolated from Freshwater Ornamental Fishes

Background: In India, canine Babesiosis has been emerged as a serious canine haemoprotozoan disease caused by protozoa namely Babesia vogeli and Babesia gibsoni which owe their sustainment and transmission to the tick vectors. Reliable, rapid and efficient molecular detection technique is necessary for choosing the effective treatment protocol and planning of control strategies against this emerging canine health problem. 
 
Methods: Babesia gibsoni and Babesia vogeli were detected by molecular means and characterized from extracted DNA of canine blood samples from 8 selected districts of Odisha, India, by an internally controlled multiplex PCR targeting the 18S rRNA gene.&nbsp; The assay was standardized and in-house validated using positive controls and random field samples (n=61) with history of tick infestation. The kappa values were analysed for diagnostic specificity and sensitivity of multiplex PCR assay for detection of naplasma. platys, B. gibsoni, Erhlichia canis, Hepatozoon canis and B. vogeli considering singleplex PCR counterparts as standard test. The PCR products found positive, were subjected to sequencing and phylogenetic analysis. The epidemiological survey (n=280) was performed using both microscopy and the standardized mPCR and risk factors were analyzed.&nbsp; 
 
Result: The mPCR assay generated amplicons of 602 bp and 489 bp corresponding to B. vogeli and B. gibsoni along with amplicon of 218 bp targeting canine &beta;-actin gene as IAC to avoid false negative result. The kappa values for diagnostic specificity and sensitivity of multiplex PCR assay in detection of B. gibsoni and B. vogeli revealed very good agreement with their singleplex PCR counterparts (n=61). The epidemiological survey (n=280) revealed 17.14% prevalence of Babesiosis with high prevalence of B. gibsoni in Khordha districts and incidence of infection were high in summer season. Presence of ticks and limited outdoor activity were found to be significantly associated with rise in incidence of Babesiosis. The Odisha isolates aligned well within the established genetic boundaries for these species, solidifying the utility of gene markers for epidemiological tracking and comparative studies.&nbsp;

Development of Multiplex PCR for Detection of Babesiosis in Dogs along with Molecular Characterization of Pathogens

<div>Background: White spot syndrome virus (WSSV), Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Enterocytozoon hepatopenaei (EHP) are widely prevalent diseases affecting cultured and wild shrimp. These diseases lead to significant production and economic losses. There have been reports of co-infection of WSSV with IHHNV, WSSV with EHP and EHP with IHHNV. Since there are no proper management and treatment methods, early and timely detection of diseases is crucial. This study aimed to develop a multiplex PCR assay for timely and accurate diagnosis of WSSV, IHHNV and EHP, which is essential for minimizing production and economic losses in the shrimp aquaculture industry. 
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Methods: This study involves the screening and confirmation of penaeid shrimp samples for diseases viz, WSSV, IHHNV and EHP, designing of PCR primers, optimization of PCR parameters such as annealing temperature, primer concentration and validation of the assay. 
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Result: The multiplex PCR assay detected WSSV, IHHNV and EHP in a single PCR reaction. Analytical sensitivity was found to be 100 pg/&micro;l of WSSV, 100 pg/&micro;l of IHHNV and 10 pg/&micro;l of EHP. Diagnostic sensitivity of the developed assay was 100%. Multiplex PCR assay developed in this study can be used for the screening of broodstock, post larvae, live food and environmental samples for early detection of WSSV, IHHNV and EHP.</div>

A Multiplex PCR Approach for Accurate Detection of Key Pathogens to Improve Disease Surveillance in Shrimp Aquaculture

Background: Aeromonas veronii is the most common bacterium responsible for diseases in freshwater fish rearing systems. Multiple factors can be involved in the virulence processes of Aeromonas bacteria. Hence, the purpose of the present investigation was to evaluate the virulence genes, antimicrobial susceptibility and pathogenicity of A. veronii isolated from freshwater fishes.&nbsp; 
 
Methods: In this investigation, we isolated A. veronii from cultured freshwater fishes, Labeo rohita and Catla catla. A. veronii was identified by bacterial staining and culture characteristics. In addition, Polymerase chain reaction (PCR) was used to evaluate the distribution of nine virulence genes including aerolysin, cytotonic enterotoxin (442, 272 bp), elastase, enolase, flagellin, lipase, serine protease and DNase.&nbsp; 
 
Result: A total of 88 A. veronii strains were isolated, which includes 56 strains from rohu and 33 strains from catla. The strains were Gram-negative, rod-shaped, motile bacteria and the colonies are yellow on blood agar. All the A. veronii strains were positive for at least one or more virulence genes tested.&nbsp;The isolates carried more virulence genes, especially in the combination of aer,alt, ela, lip, AhyB genes were found to be more virulent. Antimicrobial susceptibility to 17 antibiotics was determined and the strains of A. veronii showed 100% resistance to tetracycline, pencillin and &beta;-lactum group of antibiotics. In addition, multiple antibiotic resistance (MAR) indexes ranged from 0.24 to 0.76, suggesting that they originated from high risk contaminated zones were (W. Godavari and Nellore districts) antimicrobials are often used.&nbsp;

Detection of Virulence Genes, Antimicrobial Susceptibility and Pathogenicity of Aeromonas veronii Isolates from Labeo rohita and Catla catla

In this study, the isolation, identification and antibiotic susceptibility of bacterial agents taken from urine and sediment samples of 10 cats and 10 dogs, which were diagnosed with urinary tract infection was aimed. Each urine and sediment samples were streaked onto blood agar which contains 5 % sheep blood, Eosin Methylene Blue agar and MacConkey agar for bacterial examination. In conclusion, Staphylococcus aureus from 4 dogs, Staphylococcus epidermidis from 2 dogs, Serratia liquefaciens from 1 dog, Plesiomonas shigelloides from 1 dog, Yersinia enterocolitica from 1 cat, were isolated and identified. In conclusion of antibiogram test, 78 % of the isolates were susceptible to Amoxicillin-Clavulanic acid, 67 % of the isolates were susceptible to Ceftriaxone and Cefoperazone, 55.5 % of the isolates were susceptible to Enrofloxacin, 22.5 % of the isolates were susceptible to Lincomycin and 22 % of the isolates were susceptible to Erythromycin. The isolates were resistant to Sulfamethoxazole-Trimethoprime and Oxacillin in the ratio of 78 % and 67 % respectively. Consequently, it is concluded that the isolates obtained from dogs highlight the resistancy to antibiotics, bacterial agents play a less role in feline urinary tract infections and the appropriate antibiotic application procedure will enhance the success of therapy.

Identification of bacterial pathogens that cause urinary tract&nbsp;infections and detection of their antibiotic susceptibility

<div style="text-align: justify;">Environment is an important aspect of plant ecology and global environmental change is of major concern that is caused by natural and human activities which alter greenhouse gas concentrations in the atmosphere. The increase in concentration of greenhouse gases is foreseen to continue to raise average global temperature. Elevated concentration of carbon dioxide with increased temperature influences the plant-disease interactions. The environment has an influence over the development as well as temporal and spatial dissemination of plant diseases. The result of change in environment can either be favorable, non favorable or impartial, as these changes can either lessen, expand or have no influence on diseases as each disease may be attributed differently to these variations in accordance to an area or time of year. Variation in environmental conditions is said to be influencing plants in natural ecosystems all around the world and change in climate directly impacts crops, along with their synergy in accordance with the microbial population. The important elements governing magnification and spread of plant diseases are temperature, moisture, light and carbon dioxide concentration. Environment change causes a significant impact on germination, reproduction, sporulation, spore dispersal, along with perforation by pathogens as a vulnerable host will not be invaded by a pernicious pathogen if the environmental conditions are not facilitative for disease. The environment influences all life stages of host as well as that of a pathogen and as a result induces an opposition to various pathosystems. Resistance mechanisms of plants, including effector-triggered immunity (ETI), pattern-triggered immunity (PTI) and defence network of hormones, are particularly influenced by environmental elements. Pathogenic virulence mechanisms like fabrication of virulence proteins and toxins, and also spore germination and survival are governed by factors such as atmospheric carbon dioxide, temperature and humidity. A large number of in-vitro experiments to understand interactions between plants and pathogens rely on predetermined pathosystems by making use of ascertained environmental conditions which take into consideration relatively small part of vital plant&ndash;pathogen&ndash;environment interactions occurring in natural ecosystem. Thus, there is a need to research effectiveness of disease management strategies that is, to assess current management strategies to understand multifaceted nature of environment-pathogen interactions for production of crops that are irrepressible to environment change.</div>

Environment-Pathogen Interaction in Plant Diseases

<div>Background:&nbsp;Ticks are of great importance in transmission of various canine tick borne diseases. Several characteristics of ticks make them outstanding vectors of pathogenic agents, the wide host range and slow feeding habit along with tendency to feed on several hosts during life cycle ensures ample opportunity to acquire and transmit pathogens. 
 
Methods: This study focuses on status of canine tick vectors and molecular detection of haemoparasites in these ticks and their host, in and around Kolkata. The blood and tick samples were collected from Dog Ward, Department of Teaching Veterinary and Clinical Complex; Faculty of Veterinary and Animal Sciences, WBUAFS at Belgachia; Veterinary Clinic of Kolkata Police Dog Squad at Alipore; Veterinary Clinic of Barrackpore Police dog Squad and samples from stray dogs were also collected from inside the University campus through a period of one year (August, 2016 to July, 2017).&nbsp; 
 
Result: The tick infestation was recorded at 41% with Rhipicephalus sanguineus, being the only tick. Nine primer sets were used for detection of Babesia spp, Ehrlichia canis, Ehrlichia chaffensis, Babesia gibsoni, Hepatozoon canis, Mycoplasma haemocanis, Anaplasma platys and Theileria annae from the respective tick samples and blood sample of hosts. Tick samples were found positive for Babesia spp, Ehrlichia spp. and H. canis where as the corresponding blood samples were found positive for Babesia spp, Ehrlichia spp., Mycoplasma spp. and H. canis. This study conclusively provides evidence of high rates of incidence of haemoparasitic infection or canine tick borne diseases infection and tick infestation, with at least four haemoparasites infecting the dog population and at least one tick species (Rhipicephalus sanguineus) infesting the dogs in and around Kolkata.</div>

Incidence of Canine Tick Vectors and Molecular Detection of Haemoparasites in Vectors and Hosts

Background: Uterine infections are a leading cause of infertility and economic loss in dairy cattle, often resulting from complex polymicrobial infections. Rapid and accurate detection of the causative pathogens is essential for effective management. This study focuses on assessing the prevalence of uterine infections and developing a multiplex PCR assay for simultaneous detection of key bacterial pathogens in cattle. 
 
Methods: A total of 1,905 cattle from the Vindhyan region were screened for uterine discharge and categorized as clear with tiny flakes, cloudy, mucopurulent, purulent, or haemorrhagic. Sixty uterine samples were aseptically collected for molecular analysis. Conventional PCR was employed to detect Escherichia coli, Fusobacterium necrophorum, Trueperella pyogenes, Clostridium tertium and Histophilus somni. A multiplex PCR (mPCR) assay was subsequently developed for simultaneous detection of E. coli, C. tertium and H. somni and its sensitivity, specificity and accuracy were assessed to evaluate diagnostic performance. 
 
Result: The overall prevalence of uterine infections was 10.44%, consisting of 60.80% subclinical endometritis, 26.63% clinical endometritis and 12.56% metritis. The prevalence rates of E. coli, C. tertium and H. somni were 85%, 92.9% and 73.33%, respectively. Conventional PCR detected E. coli in 54, F. necrophorum in 01, T. pyogenes in 70, C. tertium in 44 and H. somni in 12 of the 60 uterine samples. Using mPCR, 51 samples were positive for E. coli, 44 for C. tertium and 15 for H. somni. The sensitivity, specificity and accuracy of the mPCR were 100%, 66.6% and 95% for E. coli; 100% each for C. tertium; and 80%, 100% and 95% for H. somni, respectively, indicating its high diagnostic efficiency. 
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Prevalence of Uterine Infections and Development of a Multiplex PCR Assay for their Detection in Cattle

<div>Background: Illegal migration of pigs/piglets from Myanmar to Mizoram is a common practice to meet the local demands. The migrated animals are suspected as potential carrier of various microbial pathogens. The present study was conducted on isolation, identification and molecular characterization of major bacterial pathogens (Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Haemophilus parasuis, Mycoplasma hyopneumoniae and Pasteurella multocida) in pigs illegally migrated from Myanmar to Mizoram.&nbsp; 
 
Methods: A total of 209 rectal swabs and 209 nasal swabs were collected from apparently healthy migrated pigs during October 2018 to April, 2019. All the samples were processed for PCR based detection of target bacterial species followed by isolation and identification by bacteriological techniques. The bacterial species were further confirmed by BD Phoenix automated bacterial identification system and selected virulence genes of the bacterial species were determined by specific PCR assay.&nbsp; 
 
Result: By species specific PCR, 110 samples were found to be positive for selected bacterial species, of which 20 (9.57%), 1 (0.478%), 86 (41.15%), 2 (0.956%) and 1 (0.478%) were A. pleuropneumoniae, B. bronchiseptica, H. parasuis, M. hyopneumoniae and P. multocida, respectively. A total of 52 bacterial strains were isolated and identified, of which 11, 1, 39 and 1 were A. pleuropneumoniae, B. bronchiseptica, H. parasuis, M. hyopneumoniae and P. multocida, respectively. Virulence genes were detected in A. pleuropneumoniae and H. parasuis isolates. Based upon the published literatures, this is the first ever report of isolation and identification of pathogenic A. pleuropneumoniae and H. parasuis in pigs in India.</div>