Occurrence of Coxiellosis in ruminants and its associated risk factors.

Coxiellosis in animals is caused by the zoonotic pathogen, Coxiella burnetii. Although the disease is of public health importance it remains underdiagnosed and underreported. The cross- sectional study was aimed to estimate the occurrence of the disease in livestock of study area and also to identify the risk factors associated with the disease in animals. Blood, serum, and vaginal swabs samples were collected from 200 ruminants (cattle, sheep, and goats), across various farms in Karnataka, India. These samples were then screened using ELISA and PCR (com1 and IS1111). A questionnaire was administered to the farm owners to collect the risk factor-related information. About 5.26 % cattle, 12.3 % sheep, and 12.5 % goats were positive by ELISA. By PCR, 9.47 % cattle, 9.3 % sheep, and 10 % goats were positive. Overall, the occurrence of 14.73 %, 18.46 % and 17.5 % was estimated in cattle, sheep and goat, respectively. PCR targeting the IS1111 gene detected higher number of samples as positive as compared to the com1 gene PCR. Higher number of vaginal swab samples were detected as positive as compared to blood. History of reproductive disorders (OR: 4.30; 95 %CI:1.95- 9.46), abortion (OR: 30.94; 95 %CI:6.30- 151.84) and repeat breeding (OR:11.36; 95 %CI:4.16- 30.99) were significantly associated with coxiellosis (p < 0.005). Multivariable analysis by logistic regression model analysis suggested retained abortion, repeat breeding and rearing of animal in semi-intensive system as factors significantly associated with the infection. Cultural identification of the PCR positive samples were cultured using embryonated egg propagation and cell culture techniques and positivity was confirmed in six samples. Phylogenetic analysis of the com1 and IS1111 gene revealed clustering based on similar geographic locations. The study estimated the occurrence of the disease in the study area and identified the potential risk factors.

Comparative analysis of diagnostic assays for scrub typhus: Unveiling enhanced approaches for accurate detection.

The study comparatively evaluated serological assays, namely, Weil Felix assay, and IgM ELISA with the gold-standard immunofluorescence test (IFAT) for the sensitive and specific serodiagnosis of scrub typhus infection in occupationally exposed groups of humans. A total of 78 serum samples collected from persons affected with various ailments and belonging to different risk groups were screened in the study. Out of the 78 serum samples tested, a total of 17, 26, and 47 samples turned out to be positive by IFAT, IgM ELISA, and Weil Felix test, respectively. The Weil Felix assay could not serve as an ideal test for screening scrub typhus infection owing to its poor sensitivity and specificity in comparison with IFAT. IgM-ELISA could be an initial screening test to detect scrub typhus suspected patient in limited resource settings.

Duplex real-time PCR assay with high-resolution melt analysis for the detection and quantification of Listeria species and Listeria monocytogenes in meat products.

Listeria contamination in foods of animal origin is one of the most concerning food safety issues. A duplex, SYBR green-based, real-time PCR assay was developed with high-resolution melting analysis-based differentiation of the genus Listeria and Listeria monocytogenes. The primers were designed and tested against other related foodborne pathogens. The assay was optimized for standard parameters in a non-orthogonal fashion and validated following international standards. The LODabs and LOQ of the assay were calculated to be 0.78 and 1.56 ng of the target DNA. The LODrel of the assay was found to be 1% Listeria DNA in background DNA. The assay was evaluated for applicability in artificially spiked samples, providing a 120 CFU/ml detection. The assay was validated with proficiency test samples and also with samples collected for surveillance analysis. This well-established and validated assay can be utilized as a qualitative and quantitative tool for addressing the Listeria contamination in the food safety contexts. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05695-2.

Antimicrobial Efficacy of Green Synthesized Nanosilver with Entrapped Cinnamaldehyde against Multi-Drug-Resistant Enteroaggregative Escherichia coli in Galleria mellonella.

The global emergence of antimicrobial resistance (AMR) needs no emphasis. In this study, the in vitro stability, safety, and antimicrobial efficacy of nanosilver-entrapped cinnamaldehyde (AgC) against multi-drug-resistant (MDR) strains of enteroaggregative Escherichia coli (EAEC) were investigated. Further, the in vivo antibacterial efficacy of AgC against MDR-EAEC was also assessed in Galleria mellonella larval model. In brief, UV-Vis and Fourier transform infrared (FTIR) spectroscopy confirmed effective entrapment of cinnamaldehyde with nanosilver, and the loading efficiency was estimated to be 29.50 ± 0.56%. The AgC was of crystalline form as determined by the X-ray diffractogram with a mono-dispersed spherical morphology of 9.243 ± 1.83 nm in electron microscopy. AgC exhibited a minimum inhibitory concentration (MIC) of 0.008−0.016 mg/mL and a minimum bactericidal concentration (MBC) of 0.008−0.032 mg/mL against MDR- EAEC strains. Furthermore, AgC was stable (high-end temperatures, proteases, cationic salts, pH, and host sera) and tested safe for sheep erythrocytes as well as secondary cell lines (RAW 264.7 and HEp-2) with no negative effects on the commensal gut lactobacilli. in vitro, time-kill assays revealed that MBC levels of AgC could eliminate MDR-EAEC infection in 120 min. In G. mellonella larvae, AgC (MBC values) increased survival, decreased MDR-EAEC counts (p < 0.001), had an enhanced immunomodulatory effect, and was tested safe to the host. These findings infer that entrapment enhanced the efficacy of cinnamaldehyde and AgNPs, overcoming their limitations when used individually, indicating AgC as a promising alternative antimicrobial candidate. However, further investigation in appropriate animal models is required to declare its application against MDR pathogens.

Coxiella burnetii in cattle and their human contacts in a gaushala (cattle shelter) from India and its partial com1 gene sequence-based phylogenetic analysis.

Q fever caused by Coxiella burnetii is an important zoonosis and has great public health significance. A total of 905 clinical samples from 387 cattle [serum (n = 387); vaginal swabs (n = 387); milk (n = 131)] and 59 serum samples from humans were collected from gaushala (cattle shelter) and screened for anti-C. burnetii IgG antibodies in the sera using an indirect-ELISA kit. Further, the samples were tested for C. burnetii DNA employing TaqMan real-time and conventional PCR assays targeting the com1 gene. In ELISA, 9.56% and 6.78% of animal and human sera samples were positive for anti-C. burnetii antibodies, respectively. Upon pathogen detection, 3.87% sera, 1.81% vaginal swabs, and 6.87% milk samples from cattle tested positive in TaqMan real-time PCR and 1.55% sera, 0.52% vaginal swabs, and 3.05% milk samples were found positive in conventional PCR. In humans, one serum sample was positive in both the PCR assays. The PCR positive samples (n = 12) were partially sequenced and the phylogenetic tree was constructed using com1 gene sequences (n = 42) from a different host and geographical areas. The study highlights infection of cattle and their human contacts in gaushala and identifies relationships between strains identified in the gaushala and those in other parts of the globe.

Development and comparative evaluation of droplet digital PCR and quantitative PCR for the detection and quantification of Chlamydia psittaci.

Chlamydia psittaci is a zoonotic pathogen mainly transmitted by psittacine birds and poultry. The low shedding rate of the pathogen in the apparently healthy birds and human clinical cases may result in false-negative results. In the present study, a droplet digital PCR (ddPCR) assay was developed and compared with optimized quantitative PCR (qPCR) for the detection of C. psittaci from the clinical samples. The ddPCR assay was found to be comparatively more sensitive than the qPCR, wherein the limit of detection (LOD) of ddPCR was upto 2.4 copies of the DNA template, whereas, the qPCR could detect upto 38 copies of the DNA template in the reaction mixture. Overall, the developed ddPCR assay was found to be robust, specific, and could reliably quantify up to 17.8 copies of the DNA template. Finally, the applicability of the developed ddPCR assay was tested by screening the field samples (n = 124), comprising lung tissues from dead poultry and feral birds; pooled faecal samples from the free-living birds, commercial and backyard poultry farms; pharyngeal and cloacal swabs collected from the duck farms. Of these, a total of seven samples were found to be positive by the ddPCR, whereas, three samples could be detected as positive using the qPCR. The developed ddPCR could serve as a reliable screening tool, particularly in those clinical samples wherein the shedding of C. psittaci is substantially very low.

Food safety in fisheries: Application of One Health approach.

Fisheries comprise the fastest growing sector meeting the global protein requirements. Being an affordable enterprise, it is considered a safe source of food and the muscles of healthy fishes are almost sterile. However, a multitude of hazards (biological, chemical, and environmental) can be introduced into aquaculture throughout the production and supply chain. Also, it can originate from unsuitable farming practices, environmental pollution, and socio-cultural habits prevailing in various regions. Hence, with an increasing global population and demands for aquacultural products, assessment and regulation of food safety concerns are becoming significantly evident. Ensuring safe, secure, affordable, and quality food for all in a global context is pragmatically difficult. In this context, it is quite imperative to understand the ecology and dynamics of these hazards throughout the entire production chain in a One Health approach. Here, we discuss the issues and challenges faced in the fisheries sector as a whole and the need for a One Health approach to overcome such hurdles.

Exploring Galleria mellonella larval model to evaluate antibacterial efficacy of Cecropin A (1-7)-Melittin against multi-drug resistant enteroaggregative Escherichia coli.

High throughput in vivo laboratory models is need for screening and identification of effective therapeutic agents to overcome microbial drug-resistance. This study was undertaken to evaluate in vivo antimicrobial efficacy of short-chain antimicrobial peptide- Cecropin A (1-7)-Melittin (CAMA) against three multi-drug resistant enteroaggregative Escherichia coli (MDR-EAEC) field isolates in a Galleria mellonella larval model. The minimum inhibitory concentration (MIC; 2.0 mg/L) and minimum bactericidal concentration (MBC; 4.0 mg/L) of CAMA were determined by microdilution assay. CAMA was found to be stable at high temperatures, physiological concentration of cationic salts and proteases; safe with sheep erythrocytes, secondary cell lines and commensal lactobacilli at lower MICs; and exhibited membrane permeabilization. In vitro time-kill assay revealed concentration- and time-dependent clearance of MDR-EAEC in CAMA-treated groups at 30 min. CAMA- treated G. mellonella larvae exhibited an increased survival rate, reduced MDR-EAEC counts, immunomodulatory effect and proved non-toxic which concurred with histopathological findings. CAMA exhibited either an equal or better efficacy than the tested antibiotic control, meropenem. This study highlights the possibility of G. mellonella larvae as an excellent in vivo model for investigating the host-pathogen interaction, including the efficacy of antimicrobials against MDR-EAEC strains.

Current approaches for the detection of Coxiella burnetii infection in humans and animals.

Q fever (coxiellosis), caused by Coxiella burnetii, is an emerging or re-emerging zoonotic disease of public health significance and with worldwide distribution. As a causal agent of the one among the 13 global priority zoonoses, having the infectious dose as low as one bacterium, C. burnetii has been regarded as an obligate intracellular bacterial pathogen. The agent has been classified as a Group B bioterrorism agent by the Centre for Disease Control and Prevention (CDC), and the disease is included in the World Organisation for Animal Health (OIE) list of notifiable diseases. It is mainly transmitted through airborne route in humans and animals. Isolation of C. burnetii, using standard routine laboratory culture techniques was impossible until formulation of axenic-based medium. However, it is still to be included among routinely isolated laboratory pathogen, accounting prolonged incubation period (~7 days) and requirement of specific oxygen concentration (2.5% O2). Therefore, indirect diagnostic tools have been mainly used for its diagnosis. So far serology has been mostly used for testing for C. burnetii infection. The detection of C. burnetii DNA by PCR in various clinical samples have also been widely used. The disease has remained largely under-reported, underdiagnosed and as a masked zoonosis; and therefore, needs to be explored through well-planned scientific studies for knowing its true status and likely it impact in humans and animals by employing state-of-the-art diagnostics, identifying its diverse and new host range, as well as risk factors involved in different geo-climatic, behavioural and social settings as well as risk groups. Here, we reviewed the current approaches used for the detection of C. burnetii infection in humans and animals at the population and individual level.

Apparent prevalence and risk factors of coxiellosis (Q fever) among dairy herds in India.

Coxiella burnetii is a highly infectious zoonotic pathogen infecting wide range of mammals, including humans. In the present study, a total of 711 blood samples from bovines [cattle (n = 543) and buffaloes (n = 168)] from eight farms at different geographical locations in India were screened for C. burnetii targeting the IS1111 and the com1 genes. The anti-C. burnetii antibodies in serum samples were detected using indirect-ELISA kits. Also, a total of 21 parameters pertaining to animal health and farm management were identified to assess their role as possible risk factors for coxiellosis among the targeted farms. The apparent prevalence (positive for PCR and/or ELISA) for coxiellosis was reported to be 24.5% in cattle and 8.9% in buffaloes. In cattle, the detection rate of C. burnetii employing the IS1111 gene (8.5%) was found to be significantly higher (p<0.05) as compared to the com1 (6.5%) gene. The seropositivity by ELISA was higher among cattle (17.7%) than in buffaloes (8.3%). Further, on univariable analysis of risk factors, species (cattle) (OR:3.31; 95%CI:1.88-5.82), inadequate floor spacing (OR:1.64; 95%CI:1.10-2.43), mastitis (OR:2.35, 95%CI:1.45-3.81) and reproductive disorders (OR:2.54; 95%CI:1.67-3.85) were significantly (p<0.05) having high odds for coxiellosis. The multivariable logistic regression analysis of the animal level risk factors revealed that species and age were found to be significantly associated with coxiellosis. However, since the number of screened farms is limited; further research is needed with a higher number of animals to confirm the farm level odds ratio of risk factors. Quarantine and biosecurity measures including farm hygiene operations were observed to be inadequate and also the lack of awareness about coxiellosis among the farm workers. In absence of vaccination program for coxiellosis in India, robust surveillance, farm biosecurity measures and the awareness for the disease among risk groups can play an important role in the disease prevention and subsequent transmission of the pathogen.

Molecular Investigation of the Status of Ticks on Infected Cattle for Coxiella burnetii in India.

PURPOSE: In Indian subcontinent, the epidemiological studies on the status of ticks in the transmission of Coxiella burnetii have not been explored comprehensively. The objective of the present study was to investigate the status of ticks for C. burnetii among coxiellosis positive cattle. METHODS: The present study was carried out in three locations of the northern states of India. A total of 1648 tick samples were collected from the tick infested cattle (n = 146) that were tested positive for coxiellosis by indirect serum-ELISA assay and/or the trans-PCR assay. The tick samples were screened using the trans-PCR assay targeting species-specific IS1111 transposase gene of C. burnetii. The sequencing of PCR products was planned to differentiate C. burnetii and Coxiella-like bacteria (CLB). RESULTS: The collected ticks were identified as Rhipicephalus microplus (n = 1049), Hyalomma anatolicum (n = 416), and Hyalomma spp. (n = 183). On molecular investigation, none of the collected tick samples were found to be positive for the IS1111 gene. CONCLUSION: The findings of the present study ruled out the involvement of ticks in circulation of the pathogen within the cattle population that were screened. However, extensive epidemiological studies are needed to conclusively rule out or establish the role of ticks as a competent vector for C. burnetii transmission in cattle and other hosts.

Comparison of two new in-house Latex Agglutination Tests (LATs), based on the DnaK and Com1 synthetic peptides of Coxiella burnetii, with a commercial indirect-ELISA, for sero-screening of coxiellosis in bovines.

The in-house developed DnaK and Com1 synthetic peptide-based Latex Agglutination Tests (LATs) were comparatively evaluated with commercial indirect-ELISA kit, to provide a rapid, economical and onsite field applicable test for seroscreening of coxiellosis in bovines.

Efficacy of Indolicidin, Cecropin A (1-7)-Melittin (CAMA) and Their Combination Against Biofilm-Forming Multidrug-Resistant Enteroaggregative Escherichia coli.

The present study examined the anti-biofilm efficacy of two short-chain antimicrobial peptides (AMPs), namely, indolicidin and cecropin A (1-7)-melittin (CAMA) against biofilm-forming multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC) isolates. The typical EAEC isolates re-validated by PCR and confirmed using HEp-2 cell adherence assay was subjected to antibiotic susceptibility testing to confirm its MDR status. The biofilm-forming ability of MDR-EAEC isolates was assessed by Congo red binding, microtitre plate assays and hydrophobicity index; broth microdilution technique was employed to determine minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs). The obtained MIC and MBEC values for both AMPs were evaluated alone and in combination against MDR-EAEC biofilms using crystal violet (CV) staining and confocal microscopy-based live/dead cell quantification methods. All the three MDR-EAEC strains revealed weak to strong biofilm-forming ability and were found to be electron-donating and weakly electron-accepting (hydrophobicity index). Also, highly significant (P < 0.001) time-dependent hydrodynamic growth of the three MDR-EAEC strains was observed at 48 h of incubation in Dulbecco's modified Eagle's medium (DMEM) containing 0.45% D-glucose. AMPs and their combination were able to inhibit the initial biofilm formation at 24 h and 48 h as evidenced by CV staining and confocal quantification. Further, the application of AMPs (individually and combination) against the preformed MDR-EAEC biofilms resulted in highly significant eradication (P < 0.001) at 24 h post treatment. However, significant differences were not observed between AMP treatments (individually or in combination). The AMPs seem to be an effective candidates for further investigations such as safety, stability and appropriate biofilm-forming MDR-EAEC animal models.

Development of the Com1 synthetic peptide-based Latex Agglutination Test (LAT) and its comparative evaluation with commercial indirect-ELISA for sero-screening of coxiellosis in cattle.

A novel Com1 synthetic peptide-based latex agglutination test (LAT) was developed and evaluated against commercial ELISA kit for sero-screening of coxiellosis in cattle. The developed test is economical, has field applicability and can serve as an important rapid tool for sero-screening of coxiellosis in cattle.

Virulence Potential, Biofilm Formation, and Antibiotic Susceptibility of Listeria monocytogenes Isolated from Cattle Housed in a Particular Gaushala (Cattle Shelter) and Organized Farm.

OBJECTIVES: The occurrence of Listeria monocytogenes was studied by using cultural and serological methods in cattle housed in a particular gaushala (cattle shelter) and organized dairy farm. MATERIALS AND METHODS: A total of 1201 samples from cattle comprising blood (n = 207), milk (n = 203), vaginal swabs (n = 210), and serum (n = 207) from an organized farm (n = 210) and blood (n = 100), milk (n = 74), vaginal swabs (n = 100), and serum (n = 100) from a gaushala (n = 100) were collected and analyzed for L. monocytogenes. All samples excluding serum were analyzed for isolation and identification of L. monocytogenes, while the serum samples were screened for seropositivity. The isolates were further subjected to assess their virulence potential (in vitro and in vivo), biofilm formation ability, and antibiotic susceptibility patterns. RESULTS: Four L. monocytogenes strains were isolated from the cattle; three (0.48%) from the organized farm and one (0.36%) from the gaushala. On serological screening of cattle from the organized dairy farm, 16.42% were found to be positive for antibodies against listeriolysin O, while cattle from the gaushala revealed 36% seropositivity. Furthermore, on characterization of the isolates for their pathogenic potential and biofilm-forming ability, all were found to be pathogenic by both in vitro and in vivo assays and were weak to moderate biofilm formers. The minimum inhibition concentration (MIC) of recovered isolates revealed resistance for ampicillin by two L. monocytogenes isolates (MIC >256 µg/mL), whereas three L. monocytogenes isolates were intermediately resistant (MIC >4 µg/mL) and one resistant against amoxicillin (MIC >8 µg/mL). However, all four isolates were susceptible to gentamicin, cotrimoxazole, and erythromycin. CONCLUSIONS: Isolation of virulent and antibiotic-resistant strains of L. monocytogenes warrants the need for epidemiological surveillance, antimicrobial susceptibility, and implementation of control measures to combat the occurrence of L. monocytogenes infection in animals as well as humans.

Seroprevalence and molecular detection of coxiellosis among cattle and their human contacts in an organized dairy farm.

BACKGROUND: The present investigation of Coxiella burnetii infection in cattle and farm workers on an organized cattle dairy farm, which appears to be the first of its kind in India, was undertaken to assess the status of this largely neglected and masked zoonosis. METHODS: A total of 665 samples comprising of serum (n=224), milk (n=217) and vaginal swabs (n=224) collected from milch animals (n=224) with a history of reproductive disorders were screened. Besides these, ticks (n=114); animal feed (n=4) and environmental samples (n=13) as well as serum (n=19) of farm workers were also collected. The animal sera and milk samples as well as human sera were tested for antibodies against C. burnetii by commercial ELISA kit, whereas, all the collected samples were subjected to trans-PCR targeting the IS1111 gene of C. burnetii. RESULTS: A high positivity for coxiellosis was detected in sera (29.91%) and milk (26.73%) samples of dairy cattle as well as sera from human contacts (84.21%) by ELISA. The trans-PCR detected the pathogen in 12.94% sera, 14.73% vaginal swabs and 5.53% milk samples of cattle, and in one soil sample, however, the sera of the farm workers and tick were tested negative. CONCLUSIONS: The high positivity for coxiellosis among cattle and farm workers highlight the need to undertake extensive epidemiological studies to unravel the trends of C. burnetii infection in India.

Loop-mediated isothermal amplification assay for detection of Coxiella burnetii targeting the com1 gene.

We developed the com1 gene based loop-mediated isothermal amplification (LAMP) assay for the detection of Coxiella burnetii and validated it by screening DNA isolated from serum samples collected from animals and humans. The detection of Coxiella by LAMP assay was comparable with the com1 based-PCR.

Apparent prevalence and risk factors associated with occurrence of Coxiella burnetii infection in goats and humans in Chhattisgarh and Odisha, India.

Coxiella burnetii is one of the most contagious pathogen associated with Q fever in humans, while, ruminants act as important source of infection for humans. In the present cross sectional study, a total of 464 samples were collected from 218 goats comprising of 218 sera, 218 blood and 28 milk from various parts of Chhattisgarh and Odisha region, India. Besides, environmental (33; soil- 4, faecal- 10, feed-6, drainage water- 6, drinking water- 7) and rodent (38) samples were also collected from the premises of the animals. Human sera samples (93) were collected from same sampling area comprised of workers at an organized dairy farm (43), and farmers (50). The samples were subjected to PCR targeting the trans and com1 genes and detection of antibodies using commercial ELISA kits. An overall 14.22% (95% CI: 10.2-19.47%) of the goat samples were positive using either PCR or ELISA. While, by using PCR and ELISA, 11.93% (26/218) and 9.63% (21/218) of the samples were positive for C. burnetii. A higher seropositivity (46.24%; 95% CI: 36.46-56.32%) was observed for antibodies against C. burnetii in samples collected from humans. None of the human, environmental and rodent samples were positive for C. burnetii using PCR. This seems to be the first cross sectional study to focus the hidden threat of coxiellosis among goat population and associated risk factors in India.

Listeria goaensis sp. nov.

Two Listeria-like isolates obtained from mangrove swamps in Goa, India were characterized using polyphasic combinations of phenotypic, chemotaxonomic and whole-genome sequence (WGS)-based approaches. The isolates presented as short, non-spore-forming, Gram-positive rods, that were non-motile, oxidase-negative, catalase-positive and exhibited α-haemolysis on 5 % sheep- and horse-blood agar plates. The 16S rRNA gene sequences exhibited 93.7-99.7 % nucleotide identity to other Listeria species and had less than 92 % nucleotide identity to species of closely related genera, indicating that the isolates are de facto members of the genus Listeria. Their overall fatty acid composition resembled that of other Listeria species, with quantitative differences in iso C15 : 0, anteiso C15 : 0, iso C16 : 0, C16 : 0, iso C17 : 0 and anteiso C17 : 0 fatty acid profiles. Phylogeny based on 406 core coding DNA sequences grouped these two isolates in a monophyletic clade within the genus Listeria. WGS-based average nucleotide identity and in silico DNA-DNA hybridization values were lower than the recommended cut-off values of 95 and 70 %, respectively, to the other Listeria species, indicating that they are founding members of a novel Listeria species. We suggest the name Listeriagoaensis sp. nov. be created and the type strain is ILCC801T (=KCTC 33909;=DSM 29886;=MCC 3285).

A comparative study for detection of extended spectrum ß-lactamase (ESBL) production by Enteroaggregative Escherichia coli (EAEC) strains using double disc, nitrocefin and PCR assays.

We explored and evaluated for the first time colorimetric nitrocefin assay in conjunction with the double disc test and PCR assay. We suggested the use of nitrocefin assay for rapid screening of ESBL-production by Enteroaggregative Escherichia coli.