Spatial and temporal analysis of haemorrhagic septicaemia outbreaks in India over three decades (1987-2016).

Haemorrhagic septicaemia (HS) is an economically important disease affecting cattle and buffaloes and the livelihoods of small-holder farmers that depend upon them. The disease is caused by Gram-negative bacterium, Pasteurella multocida, and is considered to be endemic in many states of India with more than 25,000 outbreaks in the past three decades. Currently, there is no national policy for control of HS in India. In this study, we analysed thirty year (1987-2016) monthly data on HS outbreaks using different statistical and mathematical methods to identify spatial variability and temporal patterns (seasonality, periodicity). There was zonal variation in the trend and seasonality of HS outbreaks. Overall, South zone reported maximum proportion of the outbreaks (70.2%), followed by East zone (7.2%), Central zone (6.4%), North zone (5.6%), West zone (5.5%) and North-East zone (4.9%). Annual state level analysis indicated that the reporting of HS outbreaks started at different years independently and there was no apparent transmission between the states. The results of the current study are useful for the policy makers to design national control programme on HS in India and implement state specific strategies. Further, our study and strategies could aid in implementation of similar approaches in HS endemic tropical countries around the world.

Pasteurella sp. associated with fatal septicaemia in six African elephants.

The sudden mortality of African elephants (Loxodonta africana) in Botswana and Zimbabwe in 2020 provoked considerable public interest and speculation. Poaching and malicious poisoning were excluded early on in the investigation. Other potential causes included environmental intoxication, infectious diseases, and increased habitat stress due to ongoing drought. Here we show evidence of the mortalities in Zimbabwe as fatal septicaemia associated with Bisgaard taxon 45, an unnamed close relative of Pasteurella multocida. We analyse elephant carcasses and environmental samples, and fail to find evidence of cyanobacterial or other intoxication. Post-mortem and histological findings suggest a bacterial septicaemia similar to haemorrhagic septicaemia caused by P. multocida. Biochemical tests and 16S rDNA analysis of six samples and genomic analysis of one sample confirm the presence of Bisgaard taxon 45. The genome sequence contains many of the canonical P. multocida virulence factors associated with a range of human and animal diseases, including the pmHAS gene for hyaluronidase associated with bovine haemorrhagic septicaemia. Our results demonstrate that Bisgaard taxon 45 is associated with a generalised, lethal infection and that African elephants are susceptible to opportunistically pathogenic Pasteurella species. This represents an important conservation concern for elephants in the largest remaining metapopulation of this endangered species.

Genomic characterization and comparative genomic analysis of HS-associated Pasteurella multocida serotype B:2 strains from Pakistan.

BACKGROUND: Haemorrhagic septicaemia (HS) is a highly fatal and predominant disease in livestock, particularly cattle and buffalo in the tropical regions of the world. Pasteurella multocida (P. multocida), serotypes B:2 and E:2, are reported to be the main causes of HS wherein serotype B:2 is more common in Asian countries including Pakistan and costs heavy financial losses every year. As yet, very little molecular and genomic information related to the HS-associated serotypes of P. multocida isolated from Pakistan is available. Therefore, this study aimed to explore the characteristics of novel bovine isolates of P. multocida serotype B:2 at the genomic level and perform comparative genomic analysis of various P. multocida strains from Pakistan to better understand the genetic basis of pathogenesis and virulence. RESULTS: To understand the genomic variability and pathogenomics, we characterized three HS-associated P. multocida serotype B:2 strains isolated from the Faisalabad (PM1), Peshawar (PM2) and Okara (PM3) districts of Punjab, Pakistan. Together with the other nine publicly available Pakistani-origin P. multocida strains and a reference strain Pm70, a comparative genomic analysis was performed. The sequenced strains were characterized as serotype B and belong to ST-122. The strains contain no plasmids; however, each strain contains at least two complete prophages. The pan-genome analysis revealed a higher number of core genes indicating a close resemblance to the studied genomes and very few genes (1%) of the core genome serve as a part of virulence, disease, and defense mechanisms. We further identified that studied P. multocida B:2 strains harbor common antibiotic resistance genes, specifically PBP3 and EF-Tu. Remarkably, the distribution of virulence factors revealed that OmpH and plpE were not present in any P. multocida B:2 strains while the presence of these antigens was reported uniformly in all serotypes of P. multocida. CONCLUSION: This study's findings indicate the absence of OmpH and PlpE in the analyzed P. multocida B:2 strains, which are known surface antigens and provide protective immunity against P. multocida infection. The availability of additional genomic data on P. multocida B:2 strains from Pakistan will facilitate the development of localized therapeutic agents and rapid diagnostic tools specifically targeting HS-associated P. multocida B:2 strains.

Reverse vaccinology-based identification of a novel surface lipoprotein that is an effective vaccine antigen against bovine infections caused by Pasteurella multocida.

Pasteurella multocida can infect a multitude of wild and domesticated animals, with infections in cattle resulting in hemorrhagic septicemia (HS) or contributing to bovine respiratory disease (BRD) complex. Current cattle vaccines against P. multocida consist of inactivated bacteria, which only offer limited and serogroup specific protection. Here, we describe a newly identified surface lipoprotein, PmSLP, that is present in nearly all annotated P. multocida strains isolated from cattle. Bovine associated variants span three of the four identified phylogenetic clusters, with PmSLP-1 and PmSLP-2 being restricted to BRD associated isolates and PmSLP-3 being restricted to isolates associated with HS. Recombinantly expressed, soluble PmSLP-1 (BRD-PmSLP) and PmSLP-3 (HS-PmSLP) vaccines were both able to provide full protection in a mouse sepsis model against the matched P. multocida strain, however no cross-protection and minimal serum IgG cross-reactivity was identified. Full protection against both challenge strains was achieved with a bivalent vaccine containing both BRD-PmSLP and HS-PmSLP, with serum IgG from immunized mice being highly reactive to both variants. Year-long stability studies with lyophilized antigen stored under various temperatures show no appreciable difference in biophysical properties or loss of efficacy in the mouse challenge model. PmSLP-1 and PmSLP-3 vaccines were each evaluated for immunogenicity in two independent cattle trials involving animals of different age ranges and breeds. In all four trials, vaccination with PmSLP resulted in an increase in antigen specific serum IgG over baseline. In a blinded cattle challenge study with a recently isolated HS strain, the matched HS-PmSLP vaccine showed strong efficacy (75-87.5% survival compared to 0% in the control group). Together, these data suggest that cattle vaccines composed of PmSLP antigens can be a practical and effective solution for preventing HS and BRD related P. multocida infections.

Viral Haemorrhagic Septicemia Virus (VHSV) Isolated from Atlantic Herring, Clupea harengus, Causes Mortality in Bath Challenge on Juvenile Herring.

Viral hemorrhagic septicaemia virus (VHSV) has been demonstrated to cause high mortalities in a wide range of teleosts, farmed as well as wild. In Europe, VHSV of genotypes Ib, Id, II, and III have been detected in wild fish, including Atlantic herring Clupea harengus, but disease outbreaks have not been observed in Atlantic herring and the effects on wild stocks are not well documented. Here, we have tested two VHSV isolates from herring (genotypes Ib and III, from the western coasts of Norway and Denmark, respectively) in a challenge experiment with herring (mean weight 2.59 g, SD 0.71 g) caught on the west coast of Denmark. The Norwegian genotype Ib isolate (NO-F-CH/2009) showed an accumulated mortality of 47% compared to 6% mortality with the Danish genotype III isolate 4p168 and zero in the unchallenged control group. In both groups, we found positive rt-RT-PCR and positive immunohistochemistry of VHSV from days 6 and 8 onward. With both isolates, the organs mainly affected were the heart and kidney. The results demonstrate the susceptibility of Atlantic herring to VHSV, and both genotypes gave pathological findings in several organs. Genotype III showed a low mortality rate, and the importance of this genotype for herring is therefore not determined. Genotype Ib showed both high prevalence and mortality, and this genotype is therefore likely to have a negative effect on wild Atlantic herring stocks. Further examinations to determine how VHSV can affect wild Atlantic herring stocks are needed.

Development of ELISA-based diagnostic methods for the detection of haemorrhagic septicaemia in animals.

Haemorrhagic septicaemia (HS) is an acute infection of cattle and buffaloes caused by the B:2 serotype of Pasteurella multocida. This disease is highly endemic in South Asia. In some peracute cases, there is 100% mortality in infected animals within a few hours of infection. Therefore, timely diagnosis of infection may contribute to its treatment and control to minimize economic losses. The current work reported the development of ELISA-based assays for the detection of anti-P. multocida antibodies and pathogen i.e. P. multocida. Owing to high immunogenicity, membrane proteins (MPs) extracted from local isolates of P. multocida serotype B:2 (PM1, PM2, and PM3) were employed as a potential diagnostic antigen for the development of indirect ELISA (i-ELISA) to detect HS antibodies in animals. MPs extracted from PM1, PM2 and PM3 isolates showed very low heterogeneity; hence MPs from the PM3 isolate were selected for the development of i-ELISA. The concentration of MPs (as coating antigen) of 3.13 µg/well and test sera dilution 1:100 was found to be optimal to perform i-ELISA. The developed method was validated through the detection of anti-P. multocida antibodies in sera of mice, immunized with MPs and formalin killed cells from the three local isolates (PM1, PM2 and PM3) of P. multocida. The significantly higher antibody titer in immunized mice was determined compared to unimmunized mice with the cut off value of 0.139. To detect P. multocida directly from the blood of infected animals, whole cell-based ELISA (cb-ELISA) assay was developed. A better detection signal was observed in the assay where bacterial cells were directly adsorbed on plate wells as compared to poly L-lysine (PLL) assisted attachment at a cell concentration of 106 CFU and 107 CFU respectively. The developed assays can be scaled up and potentially be used for the rapid detection of HS antibodies to gauge the immune status of the animal as well as vaccination efficacy and pathogen detection.

Antibiotic resistance and virulence factor gene profile of A. hydrophila isolated from carp (Cyprinidae) suspected with hemorrhagic septicemia in Gilan, Iran.

The present study was conducted to determine the antibacterial resistance profile of Aeromonas hydrophila (n = 42) isolated from the 100 hemorrhagic septicemia-suspected carp in Gilan, Iran. The prevalence of class 1 and 2 integrons, antibiotic resistance genes (ARG) and virulence factor genes (VFG) among these isolates was investigated using PCR. Also, the possible association between the presence of VFGs and the antibiotic resistance profile of isolates was assessed. The majority of A. hydrophila isolates (83·33%) exhibited multi-drug resistance (MDR) profile, and all isolates were resistant to clindamycin, while all isolates were susceptible to amikacin. intI1 and intI2 gene was found in 26·2 and 4·8% isolates, respectively. This is the first report of the presence of the intI2 gene in A. hydrophila isolates in Iran. The blaTEM (40·5%) and tetA (33·3%) genes were found as the predominant ARGs. The most frequently detected VFGs were lip and ahh1(90·5%), while the examined isolates carrying at least three VFGs and the most prevalent VFGs profile was ast+, act+, alt+, ahhl+, aerA+, ahyB+ and lip+. The results of this study indicate a positive association between the presence of VFGs and antibiotic resistance, and most MDR A. hydrophila isolates showed high frequencies of VFGs.

Efficacy of Chitosan-PLGA encapsulated trivalent oral vaccine against viral haemorrhagic septicemia virus, Streptococcus parauberis, and Miamiensis avidus in olive flounder (Paralichthys olivaceus).

The present study was conducted to assess the protective efficacy of a trivalent oral vaccine containing chitosan-PLGA encapsulated inactivated viral haemorrhagic septicemia virus (VHSV), Streptococcus parauberis serotype I and Miamiensis avidus antigens, followed by its oral (incorporated in feed) administration to olive flounder (Paralichthys olivaceus) fingerlings for a period of 15-consecutive days. After 35 days of initial vaccination, three separate challenge studies were conducted at the optimal temperature of the targeted pathogens using an intraperitoneal injection route. RPS analysis revealed moderate protection in the immunized group against all the three pathogens viz., VHSV (53.30% RPS), S. parauberis serotype-I (33.30% RPS), and M. avidus (66.75% RPS), as compared to the respective non-vaccinated challenge (NVC) control group. In addition, the immunized fish demonstrated significantly (p < 0.05) higher specific antibody titres in serum and significant (p < 0.05) upregulation in the transcript levels of immune genes of Igs (IgM, IgT, pIgR), TLRs (TLR 2, TLR 7), cytokines (IL-1ß, IL-8) and complement pathway (C3) in the mucosal and systemic tissues than those of NVC control fish, suggesting orchestration of pathogen-specific host immune responses thereby favouring its combativeness against the three pathogens. The expression dynamics of IFN-γ, Mx, caspase 3 genes post VHSV challenge; IFN-γ, TLR 2, caspase 1 genes post S. parauberis serotype I challenge and CD-8α, IL-10, TNF-α genes post M. avidus challenge further substantiates the efficacy of the vaccine in stimulating antiviral, antibacterial and antiparasitic immune responses in the host resulting in their better survival. The findings from the present study reflect that the formulated trivalent oral vaccine incorporating VHSV, S. parauberis serotype I and M. avidus antigens can be a promising prophylactic strategy to prevent the associated disease outbreaks in olive flounder.

Development of a recombinase polymerase amplification assay for viral haemorrhagic septicemia virus.

Viral diseases of fish cause significant economic losses in the aquaculture industry. Viral haemorrhagic septicemia virus (VHSV) is one of the most important viral diseases that affects more than 80 fish species. Detection of the disease, especially in the field, is critical to managing disease prevention and control programmes. Recombinase polymerase amplification (RPA) is an isothermal method with a very short amplification period and a single incubation temperature ranging from 37 to 42°C, which is a good alternative to the polymerase chain reaction (PCR). This study aimed to develop an RPA assay as sensitive as a real-time RT-PCR to detect VHSV. For this purpose, primers and probes are designed for the same targeted region of gG of VHSV. The ssRNA standards were prepared to find the detection limits of the assay. Detection limits were found ten-fold differences between real-time RT-PCR and real-time RT-RPA. While the detection limit of the RT-PCR was found as 95.5 viral RNA molecules/reaction in 95% probit value, the detection limit of RT-RPA was found as 943.75 viral RNA molecules/reaction in 95% probit value using ssRNA standards. These results show that RPA is a suitable test for VHSV Ie detection.

Comparative genome analysis of Pasteurella multocida serogroup B:2 strains causing haemorrhagic septicaemia (HS) in bovines.

Pasteurella multocida, a Gram-negative bacterium with ubiquitous nature, is known to affect wide range of host species worldwide with varied clinical manifestations including haemorrhagic septicaemia (HS) in bovines. Although, HS causing P. multocida strains were identified and characterized by conventional tools and PCR assays, diverse strains are indistinguishable by these tools in the face of disease outbreaks. In this study, draft genomes of three virulent P. multocida serotype B:2 strains (NIVEDIPm32, NIVEDIPm34 and NIVEDIPm35) were analyzed following whole genome sequencing, assembly, annotation and compared them with existing global genomes (n = 43) of bovine origin in the database. Three draft genomes of NIVEDIPm strains consisted of 40-52 contigs with GC content of ∼40.4%. The genome size and predicted genes content was ∼2.3 Mb and 2181-2189, respectively. Besides, the presence of various mobile genetic elements, antimicrobial resistance genes and biofilm related genes suggested their vital roles in virulence; further, adaptation to the host immune system as well as host pathogen interaction. Multi locus sequence analysis based on RIRDC scheme showed the presence of ST122 in all the three strains. wgMLST based phylogenic analysis suggested that HS causing Indian virulent field strains differed geographically and showed diversity from existing HS vaccine strain P52. The phylogenetic tree revealed that North Indian strains share high similarity with strains of Pakistan than South Indian Strain. Notably, a high divergence of SNPs between the HS causing circulating virulent strains of India and current HS vaccine strain P52 suggested an imminent need for relook in to HS vaccination strategy for livestock in India.

Comparative study on antigen persistence and immunoprotective efficacy of intramuscular and intraperitoneal injections of squalene - aluminium hydroxide (Sq + Al) adjuvanted viral hemorrhagic septicaemia virus vaccine in olive flounder (Paralichthys olivaceus).

The profitability of the olive flounder (Paralichthys olivaceus) aquaculture industry in Korea depends on high production and maintenance of flesh quality, as consumers prefer to eat raw flounders from aquaria and relish the raw muscles as 'sashimi'. For sustaining high production, easy-to-deliver and efficient vaccination strategies against serious pathogens, such as viral hemorrhagic septicemia virus (VHSV), is very important as it cause considerable losses to the industry. Whereas, a safe and non-invasive vaccine formulation that is free from unacceptable side-effects and does not devalue the fish is needed to maintain flesh quality. We previously developed a squalene-aluminium hydroxide (Sq + Al) adjuvanted VHSV vaccine that conferred moderate to high protection in flounder, without causing any side effects when administered through the intraperitoneal (IP) injection route. However, farmers often demand intramuscular (IM) injection vaccines as they are relatively easy to administer in small fishes. Therefore, we administered the developed vaccine via IP and IM routes and investigated the safety and persistency of the vaccine at the injection site. In addition, we conducted a comparative analysis of vaccine efficacy and serum antibody response. The clinical and histological observation of the IM and IP groups showed that our vaccine remained persistence at the injection sites for 10-17 weeks post vaccination (wpv), without causing any adverse effects to the fish. The relative percentage of survival were 100% and 71.4% for the IP group and 88.9% and 92.3% for the IM group at 3 and 17 wpv, respectively. Thus, considering the persistency period (24 wpv) and both short and long-term efficacy of our vaccine, the present study offers an option to flounder farmers in selecting either IM or IP delivery strategy according to their cultured fish size and harvesting schedule - IM vaccination for small-sized fish and IP vaccination for table-sized fish.

Changes in the behaviour of monocyte subsets in acute post-traumatic sepsis patients.

Trauma remains a major public health problem worldwide, marked as the fourth leading cause of death among all diseases. Trauma patients who survived at initial stages in the Emergency Department (ED), have significantly higher chances of mortality due to sepsis associated complications in the ICU at the later stage. There is paucity of literature regarding the role of circulating monocytes subsets and development of sepsis complications following trauma haemorrhagic shock (THS). The study was conducted to investigate the circulating level of monocyte subsets (Classical, Inflammatory, and Patrolling) and its functions in patients with acute post-traumatic sepsis. A total 72, THS patients and 30 age matched healthy controls were recruited. Blood samples were collected at different time points on days 1, 7, and 14 to measure the serum levels of cytokines by Cytometric bead assay (CBA), for the immunophenotyping of monocytes subsets, and also for the cell sorting of monocytes subsets for the functional studies. The circulating levels of monocytes subsets were found to be significantly differs among THS patients, who developed sepsis when compared with others who did not. The levels of patrolling monocytes were elevated in THS patients who developed sepsis and showed negative correlation with Sequential organ failure assessment (SOFA) score on days 7 and 14. Classical monocytes responded strongly to bacterial TLR-agonist (LPS) and produced anti-inflammatory cytokines, whereas patrolling monocytes responded with viral TLR agonist TLR-7/8 (R848) and produced inflammatory cytokines in post-traumatic sepsis patients. In conclusion, this study shows disparity in the behaviour of monocytes subsets in patients with acute post-traumatic sepsis.

Immune response in dairy cattle against combined foot and mouth disease and haemorrhagic septicemia vaccine under field conditions.

BACKGROUND: Foot-and-mouth disease (FMD) and Haemorrhagic septicemia (HS) are two important diseases that are known to have caused significant economic losses to the cattle industry. Accordingly, vaccinations have been recognized as an efficient method to control and prevent both of the above-mentioned diseases. This study aimed to determine the immune response to FMD virus antigens and the recombinant outer membrane protein of HS (rOmpH) of Pasteurella multocida in cattle administered as a combination vaccine and compare antibody titers with the two vaccines given independently, under field conditions. Dairy cattle were divided into three groups. Each group was immunized with different vaccine types according to the vaccination program employed in this study. Antibody responses were determined by indirect ELISA, liquid phase blocking ELISA (LPB-ELISA) and viral neutralization test (VNT). Furthermore, the cellular immune responses were measured by lymphocyte proliferation assay (LPA). RESULTS: The overall antibody titers to HS and FMDV were above cut-off values for the combined FMD-HS vaccine in this study.The mean antibody titer against HS after the first immunization in the combined FMD-HS vaccine groups was higher than in the HS vaccine groups. However, no statistically significant differences (p > 0.05) were observed between groups. Likewise, the antibody titer to the FMDV serotypes O/TAI/189/87 and Asia 1/TAI/85 determined by LPB-ELISA in the combined vaccine were not statistically significantly different when compared to the FMD vaccine groups. However, the mean VNT antibody titer of combined vaccine against serotype O was significantly higher than the VN titer of FMD vaccine groups (p < 0.05). Moreover, the LPA results showed that all vaccinated groups displayed significantly higher than the negative control (p < 0.05). Nevertheless, no differences in the lymphocyte responses were observed in comparisons between the groups (p > 0.05). CONCLUSIONS: The combined FMD-HS vaccine formulated in this study could result in high both antibody and cellular immune responses without antigenic competition. Therefore, the combined FMD-HS vaccine can serve as an alternative vaccine against both HS and FMD in dairy cattle under field conditions.

Protective efficacy of phage PVN02 against haemorrhagic septicaemia in striped catfish Pangasianodon hypophthalmus via oral administration.

Haemorrhagic septicaemia caused by Aeromonas hydrophila in striped catfish (Pangasianodon hypophthalmus) is one of the most important aquatic diseases in the Mekong Delta, Vietnam. However, antibiotic-resistant A. hydrophila strains have become popular and resulted in inadequate control of the disease in striped catfish farms. This study investigates the protective efficacy of bacteriophage PVN02 against haemorrhagic septicaemia in striped catfish via oral administration. The phage-containing pellets were prepared by spraying the phage solution on food pellets at 20 ml/kg. The rate of phage desorption from the food pellets into the water was very low; the phage titres in the water were approximately log 1.0 PFU/ml or undetectable. The in vivo experiment evaluating the protective efficacy of PVN02 against haemorrhagic septicaemia in striped catfish was conducted using 21 groups of 1,260 fish in 50-L plastic tanks in triplicate. The catfish were fed twice daily with phage-sprayed pellets. Different densities of bacterial suspensions were added into the tanks for 24 hr. Without the existence of the phage, the highest mortality rate was 68.3 ± 2.9% at the highest density of bacterial suspension. In contrast, the mortality rate at the highest density of bacterial suspension was significantly reduced to 8.33 ± 2.9% or 16.67 ± 2.9% at the phage dose of log 6.2 ± 0.09 or log 4.2 ± 0.09 PFU/g. This study provides a very practical manner of applying phage therapy to prevent disease in large-scale striped catfish farms.

Isolation and characterization of two virulent Aeromonads associated with haemorrhagic septicaemia and tail-rot disease in farmed climbing perch Anabas testudineus.

Diseased Anabas testudineus exhibiting signs of tail-rot and ulcerations on body were collected from a fish farm in Assam, India during the winter season (November 2018 to January 2019). Swabs from the infected body parts were streaked on sterilized nutrient agar. Two dominant bacterial colonies were obtained, which were then isolated and labelled as AM-31 and AM-05. Standard biochemical characterisation and 16S rRNA and rpoB gene sequencing identified AM-31 isolate as Aeromonas hydrophila and AM-05 as Aeromonas jandaei. Symptoms similar to that of natural infection were observed on re-infecting both bacteria to disease-free A. testudineus, which confirmed their virulence. LC50 was determined at 1.3 × 104 (A. hydrophila) and 2.5 × 104 (A. jandaei) CFU per fish in intraperitoneal injection. Further, PCR amplification of specific genes responsible for virulence (aerolysin and enterotoxin) confirmed pathogenicity of both bacteria. Histopathology of kidney and liver in the experimentally-infected fishes revealed haemorrhage, tubular degeneration and vacuolation. Antibiotic profiles were also assessed for both bacteria. To the best of our knowledge, the present work is a first report on the mortality of farmed climbing perch naturally-infected by A. hydrophila as well as A. jandaei, with no records of pathogenicity of the latter in this fish.

Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells.

MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.

Patterns of the innate immune response in tambaqui Colossoma macropomum: Modulation of gene expression in haemorrhagic septicaemia caused by Aeromonas hydrophila.

The production of tambaqui Colossoma macropomum has recently reached a milestone, being considered the main native species produced in South American continental waters. Despite the importance of this fish, its immunity is not well understood. In this study we established some patterns of innate immunity for the species via two experiments. Both studies evaluated the fish in the absence (intraperitoneal saline) or presence (intraperitoneal, 3 x 107 CFU/mL of Aeromonas hydrophila at 0.1 mL/10 g of living weight) of infection at 5 points over time-course of 14 days (0 h, 6 h, 24 h, 7 d, 14 d). In the first experiment, the partial gene sequences and gene expression of IL-1ß, IRAK-1, C3, C4, lysozyme, IL-10, HSP70 and ß-actin were determined in the main secondary lymphoid organs of fish: the spleen and head kidney. The second study was performed to analyse the alternative complement pathway ACH50 in serum to support the elucidation of C3 gene expression. Results of the gene expression assays showed a tendency towards up-regulation of immune genes in infected fish in early phases of infection (mostly around 6 h and 24 h) and in the chronic phase (7 d and 14 d), with the exception of HSP70 which showed a down-regulation in infected fish. Our results also suggested that lysozyme was evolved in both pro- and anti-inflammatory activities. For genes of the complement system, it was demonstrated that C4 regulation followed the tendency of pro-inflammatory genes. However, the C3 gene was, surprisingly, not expressed in most fish and this corroborated with the results of the complement system activity in serum that also did not show activity in most fish. The possible reasons for the regulation of gene expression and association with fish disease are addressed in this paper.

First outbreak of bovine haemorrhagic septicaemia caused by Pasteurella multocida type B in Spain - Short communication.

This paper describes the first documented outbreak of haemorrhagic septicaemia (HS) caused by Pasteurella multocida type B in cattle in Spain. This acute, highly fatal septicaemia causes major economic losses in cattle and buffaloes in many areas of Asia and Africa. In other species and in European countries it is an infrequently reported disease. Acute septicaemic pasteurellosis occurred in a free-range farm of 150 cattle and 70 beef calves in Southern Spain. Twenty-one calves and one cow were affected, of which three calves and the adult cow died. Postmortem examination revealed characteristic oedema in the ventral area of the neck and the brisket region, and widespread haemorrhages in all organs. Pure cultures of P. multocida were obtained from all tissues and organs studied. The aetiological agent was further confirmed by molecular and biochemical analysis as P. multocida capsular type B, biovar 3. Although the source of infection could not be determined, wildlife may play an important role. The use of tulathromycin in the initial stage of the disease might be related to the low morbidity and mortality of this outbreak. After using an autogenous vaccine no more cases of HS were observed.

Potential DNA Vaccine for Haemorrhagic Septiceamia Disease.

Pasteurella multocida is the main cause of haemorrhagic septicaemia (HS) outbreak in livestock, such as cattle and buffaloes. Conventional vaccines such as alum-precipitated or oil-adjuvant broth bacterins were injected subcutaneously to provide protection against HS. However, the immunity developed is only for short term and needed to be administered frequently. In our previous study, a short gene fragment from Pasteurella multocida serotype B was obtained via shotgun cloning technique and later was cloned into bacterial expression system. pQE32-ABA392 was found to possess immunogenic activity towards HS when tested in vivo in rat model. In this study, the targeted gene fragment of ABA392 was sub-cloned into a DNA expression vector pVAX1 and named as pVAX1-ABA392. The new recombinant vaccine was stable and expressed on mammalian cell lines. Serum sample collected from a group of vaccinated rats for ELISA test shows that the antibody in immunized rats was present at high titer and can be tested as a vaccine candidate with challenge in further studies. This successful recombinant vaccine is immunogenic and potentially could be used as vaccine in future against HS.

Frequência e resistência de uroculturas provenientes de pacientes internados na unidade de terapia intensiva do hospital municipal de Santarém-PA / Frequency and resistance of urocultures from patients interned in the Santarém-PA municipal hospital

Objetivo: Avaliar a frequência e a resistência dos principais microrganismos encontrados na urina de pacientes internados no hospital municipal de Santarém, Pará, nos meses de janeiro a junho de 2018. Métodos: Apresentando um estudo documental retrospectivo, descritivo, quantitativo, contrapondo as revisões bibliográficas de artigos bem esclarecidos e sucintos a respeito do tema abordado, indexadas em bases de pesquisa de artigos científicos e bibliotecas virtuais SciELO, PubMed, Lilacs e Google Acadêmico. Resultados: Dos 153 pacientes analisados, 59,6% eram mulheres, 40,4% eram homens e todos utilizavam cateter. Em relação aos uropatógenos, os mais frequentes foram os Gram-positivos: Escherichia coli com 53,8 %, Proteus mirabilis com 13,5%, juntamente com Klebsiella pneumoniae com 9,6%, Pseudomonas aeruginosa com 9,6% e Staphylococcus epidermidis com 5,8%. Foi encontrada uma prevalência de Escherichia coli de acordo com a faixa etária. Ampicilina e ciprofloxacino apresentaram respectivamente menor e maior poder inibitório contra enterobactérias. Porém, Klebsiella pneumoniae apresentou 100% de resistência às fluoquinolonas de 1ª e 2ª geração, além de apresentar padrão de resistência mais amplo. As taxas de resistência às cefalosporinas sugerem produção ESBL. Conclusão: O estudo demonstrou que o perfil dos pacientes acometidos com ITU nas UTI's era do sexo feminino (59,6%) e a bactéria mais frequente foi a Escherichia coli (53,8%). A faixa etária mais acometida foi acima de 60 anos, com 53,8% do total. Esses dados corroboram os encontrados na literatura em geral.

Objective: To evaluate the frequency and resistance of major microorganisms found in the urine of patients admitted to the municipal hospital of Santarém Pará, from January to June 2018. Methods: Presenting a retrospective, descriptive and quantitative documentary study, contrasting the bibliographic reviews of well-informed and succinct articles about the theme, indexed in scientific articles and virtual libraries research databases SciELO, PubMed, LILACS and Google Scholar. Results: Of the 153 patients analyzed, 59.6% were women and 40.4% were men, all using catheters. Gram-positive uropathogens were Escherichia coli 53.8%, Proteus mirabilis 13.5%, Klebsiella pneumoniae 9.6%, Pseudomonas aeruginosa 9.6% and Gram-positive Staphylococcus epidermidis with 5.8%. A prevalence of Escherichia coli was found according to age group. Ampicillin and ciprofloxacin showed respectively lower and higher inhibitory power against enterobacteria. However, Klebsiella pneumoniae presented 100% resistance to 1st and 2nd generation fluoquinolones, besides presenting a broader resistance pattern. Cephalosporin resistance rates suggest ESBL production. Conclusion: The study showed that the profile of patients with UTI in the ICU is female (59.6%) and the most common bacterium was Escherichia coli (53.8%). The most affected age group was over 60 years old, with 53.8% of the total. These data corroborate those found in the general literature.