Genotype diversity of brucellosis agents isolated from humans and animals in Greece based on whole-genome sequencing.

BACKGROUND: Brucellosis is a zoonotic disease whose causative agent, Brucella spp., is endemic in many countries of the Mediterranean basin, including Greece. Although the occurrence of brucellosis must be reported to the authorities, it is believed that the disease is under-reported in Greece, and knowledge about the genomic diversity of brucellae is lacking. METHODS: Thus, 44 Brucella isolates, primarily B. melitensis, collected between 1999 and 2009 from humans and small ruminants in Greece were subjected to whole genome sequencing using short-read technology. The raw reads and assembled genomes were used for in silico genotyping based on single nucleotide substitutions and alleles. Further, specific genomic regions encoding putative virulence genes were screened for characteristic nucleotide changes, which arose in different genotype lineages. RESULTS: In silico genotyping revealed that the isolates belonged to three of the known sublineages of the East Mediterranean genotype. In addition, a novel subgenotype was identified that was basal to the other East Mediterranean sublineages, comprising two Greek strains. The majority of the isolates can be assumed to be of endemic origin, as they were clustered with strains from the Western Balkans or Turkey, whereas one strain of human origin could be associated with travel to another endemic region, e.g. Portugal. Further, nucleotide substitutions in the housekeeping gene rpoB and virulence-associated genes were detected, which were characteristic of the different subgenotypes. One of the isolates originating from an aborted bovine foetus was identified as B. abortus vaccine strain RB51. CONCLUSION: The results demonstrate the existence of several distinct persistent Brucella sp. foci in Greece. To detect these and for tracing infection chains, extensive sampling initiatives are required.

Shedding rate of Brucella spp. in the milk of seropositive and seronegative dairy cattle.

Brucellosis in cattle herds has caused severe economic losses in many regions worldwide. A cross-sectional study was performed to investigate the presence of Brucella spp. in industrial dairy cattle farms in Iran. For this purpose, 935 blood and 935 milk samples were randomly collected from industrial dairy cattle farms in Iran's Alborz and Tehran provinces. Blood and milk samples were collected on the same day from each cow. Serological, bacteriological, and molecular characterization of Brucella isolates were performed using standard methods. Our results revealed the seroprevalence of brucellosis in dairy cattle farms in the Alborz and Tehran provinces, reaching 19.8%, 6.7%, 5.1%, 14.1%, and 13.1% using the Rose Bengal plate test (RBPT), serum agglutination test (SAT), 2-mercaptoethanol test (2-ME), indirect enzyme-linked immunosorbent assay (i-ELISA) and milk ring test (MRT), respectively. Furthermore, the results of bacterial culture and PCR analyses showed the presence of Brucella abortus among dairy cattle in the Alborz province and Brucella melitensis and B. abortus among dairy cattle in the Tehran province. Moreover, statistical analysis with Cohen's Kappa has highlighted the near-perfect agreement between RBPT and i-ELISA (k = 0.86). In contrast, substantial agreement was shown between RBPT and SAT performance (k = 0.70) and moderate agreement between RBPT and 2-ME (k = 0.67). The findings of this investigation showed shedding of Brucella in the milk of seropositive cows, which is a serious problem involving the maintenance and further spread of Brucella infection on the farm. Therefore, for brucellosis detection or eradication in dairy cattle farms, bacteriological and serological tests of milk samples should be performed along with blood analysis to inhibit the uncontrolled spread of the disease in animals and humans.

Spatio-Temporal Distribution of Brucellosis in European Terrestrial and Marine Wildlife Species and Its Regional Implications.

Brucellosis is an important bacterial zoonosis of domestic and wildlife species. This disease has a significant public health concern and is characterized by reproductive failure resulting in economic losses in the livestock industry. Among thirteen known species, B. abortus, B. melitensis, B. suis, and B. canis are human pathogens. Brucellosis has been extensively investigated in humans and domestic animals. However, the situation in wildlife is still not completely reported and studied. Therefore, a systematic literature search and screening were done to clarify the situation of brucellosis in wildlife in Europe. Sixty-five articles from a total of 13,424 reports published between 1991 and 2021 were selected, applying defined inclusion criteria. Wild boars and brown hares were the most often studied terrestrial wildlife species, whereas seals and porpoises were the most often investigated marine wildlife. Poland, Croatia, and Belgium showed the highest seroprevalences of wild boars caused by B. suis biovar 2. In marine wildlife, brucellosis was mainly caused by B. ceti and B. pinnipedialis. Most samples were from carcasses. Thus, sera could not be collected. It is worrisome that B.abortus and B. melitensis were reported from both terrestrial and marine wild animals, posing a zoonotic threat to people exposed to wild animals. Currently, there is no approved vaccine available for wild animals. The main challenges are the development of specific diagnostics and their validation for use in wildlife.

Q Fever-A Neglected Zoonosis.

Q fever remains a neglected zoonosis in many developing countries including Pakistan. The causing agent Coxiella (C.) burnetii is resistant to environmental factors (such as drying, heat and many disinfectants), resulting in a long-lasting infection risk for both human and animals. As the infection is usually asymptomatic, it mostly remains undiagnosed in animals until and unless adverse pregnancy outcomes occur in a herd. In humans, the infection leads to severe endocarditis and vascular infection in chronic cases. Limited data are available on molecular epidemiology and evolution of this pathogen, especially in ruminants. Genomic studies will help speculating outbreak relationships in this scenario. Likewise, pathogenesis of C. burnetii needs to be explored by molecular studies. Awareness programs and ensuring pasteurization of the dairy milk before human consumption would help preventing Q fever zoonosis.

Seroprevalence and Molecular Evidence of Coxiella burnetii in Dromedary Camels of Pakistan.

Coxiellosis is a zoonosis in animals caused by Coxiella burnetii. A cross-sectional study was conducted on 920 (591 female and 329 male) randomly selected camels (Camelus dromedarius) of different age groups from 13 districts representative of the three different ecological zones in the Province Punjab, Pakistan to determine the prevalence and associated risk factors of coxiellosis. The blood samples were collected and tested for anti-C. burnetti antibodies using indirect multispecies ELISA. Real-time PCR was used for the detection of C. burnetii DNA to determine the prevalence in heparinized blood pools. Out of 920 investigated camels, anti-C. burnetii antibodies were detected in 288 samples (31.3%) (95% CI: 28.3-34.4%). The highest (78.6%) and lowest (1.8%) seroprevalence were detected in Rahimyar Khan (southern Punjab) and in Jhang (central Punjab), respectively. Potential risk factors associated with seropositivity of the Q fever in camels included desert area (42.5%; OR = 2.78, 95% CI 1.12-3.21) summer season (35.7%; OR = 2.3, 95% CI: 1.31-3.2), sex (female) (39.1; OR = 2.35, 95% CI: 1.34-2.98), tick infestation (51.3%;OR = 2.81, 95% CI: 1.34-3.02), age (>10 years; 46.4%; OR = 1.56, 95% CI: 0.33-2.05) and herd size (38.5%; OR = 1.21, 95% CI: 0.76-1.54). Coxiella burnetii DNA was amplified in 12 (20%) and 1 (10%) of 60 ELISA-negative and 10 suspected camels, respectively. DNA could not be detected in ELISA positive blood pools. This study emphasizes the seroprevalence and associated risk factors of coxiellosis as well as its potential to spill over to animals and humans in contact with these camel herds.

Rabies Virus Exploits Cytoskeleton Network to Cause Early Disease Progression and Cellular Dysfunction.

Rabies virus (RABV) is a cunning neurotropic pathogen and causes top priority neglected tropical diseases in the developing world. The genome of RABV consists of nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase L protein (L), respectively. The virus causes neuronal dysfunction instead of neuronal cell death by deregulating the polymerization of the actin and microtubule cytoskeleton and subverts the associated binding and motor proteins for efficient viral progression. These binding proteins mainly maintain neuronal structure, morphology, synaptic integrity, and complex neurophysiological pathways. However, much of the exact mechanism of the viral-cytoskeleton interaction is yet unclear because several binding proteins of the actin-microtubule cytoskeleton are involved in multifaceted pathways to influence the retrograde and anterograde axonal transport of RABV. In this review, all the available scientific results regarding cytoskeleton elements and their possible interactions with RABV have been collected through systematic methodology, and thereby interpreted to explain sneaky features of RABV. The aim is to envisage the pathogenesis of RABV to understand further steps of RABV progression inside the cells. RABV interacts in a number of ways with the cell cytoskeleton to produce degenerative changes in the biochemical and neuropathological trails of neurons and other cell types. Briefly, RABV changes the gene expression of essential cytoskeleton related proteins, depolymerizes actin and microtubules, coordinates the synthesis of inclusion bodies, manipulates microtubules and associated motors proteins, and uses actin for clathrin-mediated entry in different cells. Most importantly, the P is the most intricate protein of RABV that performs complex functions. It artfully operates the dynein motor protein along the tracks of microtubules to assist the replication, transcription, and transport of RABV until its egress from the cell. New remedial insights at subcellular levels are needed to counteract the destabilization of the cytoskeleton under RABV infection to stop its life cycle.

Ameliorative Effects of Anti-Clostridial Egg Yolk Antibodies (IgYs) in Experimentally-Induced Avian Necrotic Enteritis.

The present study was planned to evaluate the ameliorative effects of egg yolk antibodies (EYAs) in broiler chicken. For this purpose, 80-day-old broiler chickens were divided into four groups (A-D), where group A was kept as negative control. Experimental infection with C. perfringens (1 × 108 cfu/mL) was induced via oral route on days 17, 18 and 19 of the experiment in groups B, C and D. Groups C and D were passively immunized by anti-clostridial IgYs @ 1 mL per bird via oral and oral and intramuscular (I/M) routes respectively, on days 21 to 24, and on days 22 and 24 of the experiment, respectively. Two necropsies were performed (the first on day 26th and the second on day 35th). Birds in group B showed behavioral signs e.g., laziness, depression and diarrhea, gross post-mortem lesions e.g., increase in the relative weights (RW), due to acute swelling and congestion of liver and kidneys and ballooning and hemorrhages of jejunum and microscopic lesions e.g., congestion and necrosis in liver and kidneys' parenchyma and disrupted epithelium with fewer goblet cells in jejunum, compared to the group A. Birds in groups C and D, showed significant improvements in clinical and behavioral signs, RW of liver, kidneys and jejunum, swelling, congestion and mononuclear cells' infiltration in liver and kidneys and damages in the jejunal-wall, compared to group B. The most significant changes were found in birds of group C. Our study revealed ameliorative effects of EYAs on certain biological parameters however, further studies would be needed to justify a safer production and a reliable application of EYAs in NE outbreaks.

Efficacy Evaluation of a Combined Hemorrhagic Septicemia-Mastitis Vaccine in Dairy Cows and Buffaloes.

Hemorrhagic septicemia (HS) and mastitis caused by Pasteurella (P.) multocida, Staphylococcus (S.) aureus and Streptococcus (Str.) agalactiae are important ailments of the dairy industry especially in South Asia. The present study evaluates the efficacy of a locally prepared hemorrhagic septicemia (HS) and mastitis combined vaccine. To this end, a total of 70 HS, S. aureus and Str. agalactiae-free lactating (early stage of lactation) buffaloes (n = 45) and cows (n = 25), and 50 lactating (early stage of lactation) cows (n = 25) and buffaloes (n = 25) positive for S. aureus/Str. agalactiae were subjected to two doses of HS−mastitis combined vaccine with an interval of 21 days. Antibody response was monitored by ELISA up to six months (180 days). Antibody titers against HS and mastitis were significantly (p ˂ 0.05) higher in vaccinated groups as compared to the non-vaccinated groups. Cumulative mean somatic cell counts and mastitis severity scores in vaccinated groups were significantly lower (p < 0.05), and milk yield was significantly higher (p < 0.05) than those in the respective non-vaccinated controls. In conclusion, Montanide®-adjuvanted HS−mastitis combined vaccine showed significant immunogenic effects in dairy cows and buffaloes. However, challenge studies remain overdue.

Water Purification through a Novel Electrospun Carbon Nanofiber Membrane.

Here, we report water purification through novel polyvinyl alcohol (PVA)-based carbon nanofibers synthesized through the electrospinning technique. In our novel approach, we mix PVA and tetraethyl orthosilicate (TEOS) with green tea solutions with different concentrations to synthesize carbon-based nanofibers (CNFs) and further calcine at 280 °C for carbonization. The scanning electron microscopy (SEM) results show the diameter of the nanofibers to be ∼500 nm, which decreases by about 50% after carbonization, making them more suitable candidates for the filtration process. Next, using these carbon nanofibers, we prepare filters for water purification. The synthesized CNF filters show excellent performance and successful removal of contaminants from the water by analyzing the CNF-based filters before and after the filtration of water through SEM and energy-dispersive X-ray (EDX) spectroscopy. Our SEM and EDX results indicate the presence of various nanoparticles consisting of different elements such as Mg, Na, Ti, S, Si, and Fe on the filters, after the filtration of water. Additionally, the SEM results show that PVA and TEOS concentrations play an important role in the formation, uniformity, homogeneity, and particularly in the reduction of the nanofiber diameter.

Animal and Human Brucellosis in Pakistan.

Brucellosis is a bacterial zoonotic disease that affects many animal species and can be transmitted to humans via direct contact or via contaminated food. Although brucellosis is a serious health hazard, its public health concern has been neglected in many countries. In some developing countries, such as Pakistan, where brucellosis is endemic, this disease continues to be of importance. A literature search for the past 11 years (2011-2021) provided a comprehensive insight into brucellosis in Pakistan. In this review, particular emphasis was placed on occurrence, diagnostic tests used, and prevention, treatment, and control in the context of the "One Health" approach.

Seroprevalence and Associated Risk Factors of Bovine Brucellosis in District Gujranwala, Punjab, Pakistan.

Bovine brucellosis is a contagious zoonotic disease that causes economic losses through abortion and infertility. A cross-sectional study was designed to determine the seroprevalence and associated risk factors of bovine brucellosis in district Gujranwala of Punjab, Pakistan. A total of 220 bovine sera (112 from buffaloes, 108 from cattle) from 46 unvaccinated herds were collected. Parallel testing by the Rose Bengal Plate Test (RBPT) and Indirect Enzyme-linked Immunosorbent Assay (I-ELISA) showed a 58.7% (27/46) herd-level and 22.7% (50/220) animal-level seroprevalence. Seroprevalence was higher (p < 0.001, OR = 7.62) in adult animals (37.2%) compared to younger animals (4.9%). A herd size of >10 animals (p = 0.021, OR = 7.83), less housing space (p = 0.037, OR = 6.39) and history of abortion at the farm (p = 0.023, OR = 5.6) were found as risk factors associated with the seropositivity of brucellosis. There was a substantial agreement between the RBPT and I-ELISA results (Cohen's kappa coefficient (κ) = 64.16, percent agreement = 89.5%). In conclusion, a relatively higher seroprevalence was found compared to the previous reports from the country. Standardization and validation of the advanced diagnostic tests would be needed. Biosecurity, personal protection, quarantine measures and routine screening of animals at the farm level and disease awareness programs and consumption of pasteurized milk in the human population will be helpful in preventing the transmission/zoonosis of the disease.

Characterization of fungal microbial diversity in healthy and diarrheal Tibetan piglets.

BACKGROUND: Diarrhea is an important ailment limiting the production of the Tibetan pig industry. Dynamic balance of the intestinal microbiota is important for the physiology of the animal. The objective of this work was to study fungal diversity in the feces of early weaning Tibetan piglets in different health conditions. RESULTS: In the present study, we performed high-throughput sequencing to characterize the fungal microbial diversity in healthy, diarrheal and treated Tibetan piglets at the Tibet Autonomous Region of the People's Republic of China. The four alpha diversity indices (Chao1, ACE, Shannon and Simpson) revealed no significant differences in the richness across the different groups (P > 0.05). In all samples, the predominant fungal phyla were Ascomycota, Basidiomycota and Rozellomycota. Moreover, the healthy piglets showed a higher abundance of Ascomycota than the treated ones with a decreased level of Basidiomycota. One phylum (Rozellomycota) showed higher abundance in the diarrheal piglets than in the treated. At genus level, compared with that to the healthy group, the proportion of Derxomyces and Lecanicillium decreased, whereas that of Cortinarius and Kazachstania increased in the diarrheal group. The relative abundances of Derxomyces, Phyllozyma and Hydnum were higher in treated piglets than in the diarrheal ones. CONCLUSIONS: A decreased relative abundance of beneficial fungi (e.g. Derxomyces and Lecanicillium) may cause diarrhea in the early-weaned Tibetan piglets. Addition of probiotics into the feed may prevent diarrhea at this stage. This study presented the fungal diversity in healthy, diarrheal and treated early-weaned Tibetan piglets.

The marvelous optical performance of AlGaN-based deep ultraviolet light-emitting diodes with AlInGaN-based last quantum barrier and step electron blocking layer.

The optoelectronic characteristics of AlGaN-based deep ultraviolet light-emitting diodes (DUV LEDs) with quaternary last quantum barrier (QLQB) and step-graded electron blocking layer (EBL) are investigated numerically. The results show that the internal quantum efficiency (IQE) and radiative recombination rate are remarkably improved with AlInGaN step-graded EBL and QLQB as compared to conventional or ternary AlGaN EBL and last quantum barrier (LQB). This significant improvement is assigned to the optimal recombination of electron-hole pairs in the multiple quantum wells (MQWs). It is due to the decrease in strain and lattice mismatch between the epi-layers which alleviates the effective potential barrier height of the conduction band and suppressed the electron leakage without affecting the holes transportation to the active region. Moreover, to figure out quantitatively, the electron and hole quantity increased by ~ 25% and ~ 15%, respectively. Additionally, the IQE and radiative recombination rate are enhanced by 48% and 55%, respectively, as compared to conventional LED. So, we believe that our proposed structure is not only a feasible approach for achieving highly efficient DUV LEDs, but the device physics presented in this study establishes a fruitful understanding of III nitride-based optoelectronic devices.

Environmental fluoride exposure disrupts the intestinal structure and gut microbial composition in ducks.

Fluorine (F) and its compounds produced from industrial production and coal combustion can cause air, water and soil contamination, which can accumulate in animals, plants and humans via food chain threatening public health. Fluoride exposure affects liver, kidney, gastrointestinal and reproductive system in humans and animals. Literature regarding fluoride influence on intestinal structure and microbiota composition in ducks is scarce. This study was designed to investigate these effects by using simple and electron microscopy and 16S rRNA sequencing techniques. Results indicated an impaired structure with reduced relative distribution of goblet cells in the fluoride exposed group. Moreover, the gut microbiota showed a significant decrease in alpha diversity. Proteobacteria, Firmicutes and Bacteroidetes were the most abundant phyla in both control and fluoride-exposed groups. Specifically, fluoride exposure resulted in a significant decrease in the relative abundance of 9 bacterial phyla and 15 bacterial genera. Among them, 4 phyla (Latescibacteria, Dependentiae, Zixibacteria and Fibrobacteres) and 4 genera (Thauera, Hydrogenophaga, Reyranella and Arenimonas) weren't even detectable in the gut microbiota of the ducks. In summary, higher fluoride exposure can significantly damage the intestinal structure and gut microbial composition in ducks.

Revisiting Brucellosis in Small Ruminants of Western Border Areas in Pakistan.

Brucellosis, globally known bacterial zoonosis, is endemic to Pakistan. B. abortus in bovines, B. melitensis in small ruminants and B. canis in dogs mainly cause this disease. A total of 1821 sera (1196 from sheep and 625 from goats) from animal herds near the Pakistan-Afghanistan border were collected. In parallel testing of sera for anti-Brucella antibodies (B. abortus and B. melitensis) was carried out by RBPT and indirect ELISA. The presence of Brucella DNA in sera was tested by real-time PCR. The overall percentage of seropositive samples was 0.99 (18/1821) by both tests. All positive samples originated from Baluchistan territory which translated into 1.76% (18/1021). None of the positive sera had signals for Brucella DNA and none of sera from goats carried detectable antibodies. Both tests showed an almost perfect agreement with Kappa statistics. The flock size was found to be associated with the presence of anti-Brucella antibodies. The samples of Khyber Pakhtunkhwa (KPK) tested negative in both serological tests and hence were not processed for real-time PCR. The present study shows the presence of anti-Brucella antibodies in sheep in the Baluchistan region of Pakistan. Diagnostic services need to be improved and test and slaughter policies might be implemented for eradication of Brucella infection in these areas. Awareness about the infection is needed at the farmer's level. Isolation and molecular biology of the isolates could help with understanding the prevailing etiology in a better way.

Epidemiology and Associated Risk Factors for Brucellosis in Small Ruminants Kept at Institutional Livestock Farms in Punjab, Pakistan.

Brucellosis is reportedly endemic in ruminants in Pakistan. Both Brucella abortus and B. melitensis infections have been decumented in domestic animals and humans in the country. This study aimed to identify the burden of anti-Brucella antibodies in small ruminants as well as associated potential risk factors with its occurrence at nine institutional livestock farms in Punjab, Pakistan. The sera collected from equal number of sheep and goats (500 from each species) were screened by indirect-ELISA for anti-smooth-Brucella antibodies followed by a serial detection by real-time PCR. Overall, 5.1% (51/1000) seropositivity was registered corresponding to 5% (25/500) prevalence in goats and 5.2% (26/500) in sheep. Brucella-DNA could not be detected in any of the tested sera by real-time PCR. Multiple logistic regression model indicated that farm location (OR 34.05), >4 years of age (OR 2.88), with history of reproductive disorders (OR 2.69), and with BCS of ≤ 3 (OR 12.37) were more likely to test positive for brucellosis at these farms. A routine screening, stringent biosecurity, and quarantine measures are warranted for monitoring and eradication of the infection. Similarly, isolation and molecular investigation of the etiologic agent(s) are needed to understand the relationship of epidemiology and out-breaks of brucellosis in the country.

Serological and Molecular Investigation of Brucellosis in Breeding Equids in Pakistani Punjab.

Brucellosis is an important zoonosis worldwide. Equines are susceptible to the infection when in close contact with infected animals. The objective of our study was to update the existing knowledge and detect and differentiate the causative agent of brucellosis in breeding equines in Punjab, Pakistan. A cross-sectional study was designed to evaluate the occurrence and etiology of the infection in the equine population in three districts. A total of 448 equine sera were collected from three prefectures viz. Sahiwal, Khanewal, and Okara of the Punjab Province of Pakistan. Ninety-six (21.4%) samples were found positive by RBPT, 3.56% (16/448) by iELISA, and 4.24% (19/448) by CFT. Real-time PCR demonstrated the presence of Brucella abortus-DNA in sero-positive samples. Age and location were found as risk factors. The study concludes equine brucellosis seroprevalence in the country where Brucella abortus as the main etiology. Fistulous withers and poll evil cases should be treated with care as they could be hazardous and a source of zoonotic transmission. Routine screening at an early age, vaccination in ruminants, and consumption of pasteurized dairy milk in humans is recommended for prevention of the infection. Specific tests need to be standardized and validated.

Serological and Molecular Detection of Bovine Brucellosis at Institutional Livestock Farms in Punjab, Pakistan.

Bovine brucellosis remains a persistent infection in ruminants in Pakistan. A total of 828 (409 buffaloes and 419 cattle) sera were collected from 11 institutional-owned livestock farms in Punjab, Pakistan. The samples were tested by rose bengal plate agglutination test (RBPT) and indirect enzyme-linked immunosorbent assay (iELISA). The seroprevalence along with 95% confidence interval (CI) was determined. Univariable and multivariable analysis of the epidemiological background data was conducted and odds ratio (OR) was calculated to understand any association between the risk factors and the seroprevalence. An overall seroprevalence of 3.9% (Positive/Tested = 32/828) and 3.3% (27/828) was detected by RBPT and iELISA, respectively. The seroprevalence of 5.6% (CI 3.6-8.3) and 4.7%, (CI 2.8-7.2) and the odds ratio of 2.63 (CI 1.20-5.77) and 2.50 (CI 1.08-5.78) for testing positive by RBPT and iELISA, respectively were significantly higher (p < 0.05) in buffaloes than in cattle. Breed, sex, history of abortion and retention of fetal membranes (RFM) in the animals were not found statistically significantly associated with the infection. RBPT and iELISA based results agreed almost perfect (k = 0.877). In total, Brucella abortus-DNA (9/27) was amplified from seropositive samples by real-time polymerase chain reaction. This study identified for the first time the etiological agents of brucellosis at a molecular level at institutional-owned livestock farms in Pakistan.

Crimean-Congo haemorrhagic fever virus in ticks collected from livestock in Balochistan, Pakistan.

BACKGROUND: Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne zoonotic pathogen. It causes a fatal haemorrhagic disease in humans. Hard ticks, in particular Hyalomma spp., are considered to function as reservoir as well as vector for CCHFV. METHODS: A cross-sectional study was conducted in the province of Balochistan, Pakistan, from September to November 2017. Ticks were collected from cattle, sheep and goats in livestock farms. The ticks were morphologically identified, followed by confirmation with molecular methods (PCR and sequencing). Furthermore, ticks were examined for CCHFV genomes (S segment) by a one-step multiplex real-time RT-qPCR and positive samples were sequenced to determine the CCHFV genotype. RESULTS: In total, 525 of 529 livestock infesting adult ticks belonged to the genus Hyalomma, and 4 ticks to the genus Rhipicephalus (R. microplus 3×, R. turanicus 1×). In the genus Hyalomma, H. marginatum (28%), H. excavatum (26%), H. dromedarii (22%), H. anatolicum (16%) and H. scupense (8%) ticks were identified. Tick infestations were as follows: sheep 58%, goats 28% and cattle 14%. Four per cent (20/525) of ticks were CCHFV genome-positive, and all genomes clustered in CCHFV genotype Asia 1. Among CCHFV-positive ticks, 75% (15/20) were female and 25% (5/20) male. CCHFV genomes were most frequently detected in H. marginatum (30%, 6/20), followed by H. dromedarii (25%, 5/20), H. excavatum (20%, 4/20), H. anatolicum (20%, 4/20) and H. scupense (5%, 1/20). All CCHFV-positive ticks were found on sheep. The largest number of CCHFV-positive ticks were detected in the district of Kalat (60%, 12/20), followed by the districts of Quetta (30%, 6/20) and Killa Abdullah (10%, 2/20). CONCLUSIONS: This study confirms the circulation of CCHFV in ticks in Balochistan, south-western Pakistan. It is imperative to take effective tick control measures in this area, especially to control livestock tick infestations to prevent CCHF infections in humans.

Intraperitoneal Bupivacaine as Post-laparoscopic Cholecystectomy Analgesia.

OBJECTIVE: To assess the efficacy of large volumes of diluted intraperitoneal bupivacaine in post-laparoscopic cholecystectomy analgesia. STUDY DESIGN: A randomised controlled trial. PLACE AND DURATION OF STUDY: Department of General Surgery, Nishtar Hospital, Multan, from August 2018 to June, 2019. METHODOLOGY: Two equal groups with 55 patients each were formed. Normal saline 500 ml in group A, and mixture of 20 ml 0.5% bupivacaine in 480 ml normal saline in group B, was used to irrigate peritoneal cavity. Final outcome of the study was the comparison of pain-free duration. Postoperatively, numerical rating scale (NRS) score at various intervals and total analgesics requirement within 24 hours after the procedure were included in the secondary outcomes. Student's t-test was applied on continuous data and Pearson's Chi-square test on nominal variables. P >0.05 was considered of no statistical significance. RESULTS: Both groups were comparable for age, weight, gender, duration of surgery. Postoperative analgesia duration was 0.99 ± 0.51 hours in group A and 16.53 ±2.65 hours in group-B (p<0.001). On average, 124.80 ±26.68 mg and 31.00 ±14.98 mg tramadol was given to group A and B patients, respectively (p<0.001). There was statistically significant difference in NRS score at 30 minutes, 1, 3, 6 and 12 hours postoperatively (p<0.05). NRS score at ETT extubation and at 24 hours was statistically not different (p >0.05). CONCLUSION: Large volume of diluted bupivacaine when injected intraperitoneally during laparoscopic cholecystectomy provides prolonged time pain relief.