Salmonella from Farm to Table: Isolation, Characterization, and Antimicrobial Resistance of Salmonella from Commercial Broiler Supply Chain and Its Environment.

Antimicrobial resistance (AMR) in poultry production chain is one of the major food safety concerns due to indiscriminate usage of antibiotics and the presence of pathogens such as Salmonella which causes infections in various stages of production. In the present study, 182 samples were collected from commercial broiler supply chain, viz., three hatcheries (n = 29), three commercial broiler farms (CBF; n = 99), and three retail meat shops (RMS; n = 54), and used for isolation and identification of Salmonella using three different selective agar media and a selective enrichment medium followed by PCR confirmation targeting the hilA gene. The overall prevalence of Salmonella was 47/182 (25.82%), and a significantly higher (P < 0.05) prevalence was observed in retail meat shops (46.29%), CBF (19.19%), and hatcheries (10.34%). Comparison of three agar media for isolation of Salmonella revealed that all the media were equally selective. However, PCR amplification of hilA gene fragment was significantly higher (P < 0.01) in selective enrichment culture tetrathionate brilliant green bile broth (TTB) as compared to all solid (agar-based) media. Susceptibility pattern against most frequently used antibiotics revealed that 100% of the isolates were resistant to at least one antibiotic. High resistance was observed for doxycycline (94.34%), followed by cefpodoxime (84.91%), ciprofloxacin (72.64%), gentamicin (65.09%), enrofloxacin (61.32%), colistin sulphate (40.42%), amikacin (34.91%), ampicillin (33.96%), neomycin (33.02), cefotaxime (30.19%), ceftazidime (29.25%), trimethoprim-sulfamethoxazole (23.58%), amoxicillin+clavulanic acid (21.70%), and chloramphenicol (12.26%); 16.98% of the isolates were ex-tended spectrum ß-lactamase (ESBL) producers, and 76.41% were multidrug resistant (MDR). MDR Salmonella were significantly higher (P < 0.01) in RMS (91.66%) followed by CBF (82.75%), whereas no MDR isolates were present in the isolates from hatcheries. The results indicated a higher prevalence of Salmonella and AMR for commonly used antibiotics in the complete broiler supply chain, especially RMS and CBF. Also, this study idicated that TTB enrichment followed by PCR and colony PCR was found to be rapid, specific and time-saving method.

Rabies on the Indian subcontinent.

The Indian subcontinent comprises Afghanistan, Bangladesh, Bhutan, India, Nepal, the Maldives, Myanmar, Pakistan and Sri Lanka. In all of these countries, except the Maldives, rabies is endemic. An estimated 59,000 people die from rabies each year; 45% of these deaths occur on the Indian subcontinent and approximately 33% take place in India. The majority of these deaths are attributable to dog bites, and those most affected are children and the poor. Access to post-exposure prophylaxis is limited and costly, the supply of immunoglobulins and vaccines can be irregular and public awareness of rabies is low. Moreover, the vaccination of domestic dogs is not widely implemented. There is a need for increased laboratory capacity and expertise across the continent, as well as better data, improved surveillance and more user-friendly and economical diagnostic tests. An animal birth control programme has met with mixed success in India. However, a greater focus on mass dog vaccination could eliminate the disease at its source, reducing the large burden of mortality for at-risk communities. In this paper, the authors examine the situation in each of the countries on the Indian subcontinent, discuss current needs, obstacles and progress, and examine future strategies, with the objective of eliminating dog-mediated rabies from the subcontinent by 2030.

Le sous-continent indien comprend l'Afghanistan, le Bangladesh, le Bhoutan, l'Inde, les Maldives, le Myanmar, le Népal, le Pakistan et Sri Lanka. La rage est présente à l'état endémique dans tous ces pays sauf aux Maldives. Chaque année, le nombre estimé de victimes humaines de la rage s'élève à 59 000 personnes dans le monde, dont 45 % dans le sous-continent indien et 33 % en Inde. Ces décès sont dans leur majorité associés à une morsure de chien enragé et surviennent surtout parmi les enfants et les populations pauvres. La prophylaxie post-exposition est peu accessible et coûte cher ; par ailleurs, la fourniture d'immunoglobulines et de vaccins n'est pas assurée de manière régulière et la population est peu sensibilisée au problème de la rage. En outre, la vaccination des chiens domestiques n'est pas une pratique répandue. Il est impératif d'améliorer les capacités et l'expertise technique des laboratoires du sous-continent ainsi que la qualité des données, les méthodes de surveillance et l'accès à des tests diagnostiques faciles d'emploi et peu onéreux. Un programme de stérilisation animale appliqué en Inde a eu un succès mitigé. Toutefois, une intensification des efforts visant la vaccination systématique des chiens permettrait d'éliminer la maladie à sa source, réduisant ainsi le fardeau de la mortalité par rage dans les communautés les plus exposées au risque. Les auteurs font le point sur la situation de la rage dans chaque pays du sous-continent indien en décrivant les besoins actuels, les obstacles rencontrés, les progrès enregistrés et les stratégies futures en vue d'éliminer la rage humaine transmise par les chiens du sous-continent d'ici 2030.

El subcontinente indio engloba Afganistán, Bangladesh, Bután, India, Nepal, las Maldivas, Myanmar, el Pakistán y Sri Lanka, países todos ellos, salvo las Maldivas, en que la rabia es endémica. Se calcula que la enfermedad causa la muerte de 59 000 personas al año, de las que un 45% fallecen en el subcontinente indio y aproximadamente un 33% en la India. La mayoría de estas muertes son atribuibles a mordeduras de perro, y los colectivos más afectados son los niños y las personas pobres. El acceso a medidas de profilaxis tras la exposición es deficiente y costoso, el suministro de inmunoglobulinas y vacunas puede ser irregular y la población no sabe gran cosa de la rabia. Además, la vacunación de los perros domésticos no es práctica generalizada. El subcontinente necesita más conocimientos técnicos y mayor capacidad de laboratorio, así como datos de mejor calidad, una vigilancia más eficaz y pruebas de diagnóstico más económicas y fáciles de aplicar. En la India se aplicó con resultados desiguales un programa de control de la natalidad animal. No obstante, si se hiciera mayor hincapié en la vacunación masiva de perros sería posible eliminar la enfermedad en su origen, lo que reduciría la gran carga de mortalidad que impone a las comunidades expuestas. Los autores pasan revista a la situación en cada uno de los países del subcontinente indio, exponen el conjunto de necesidades, obstáculos y avances que se observan en ellos y examinan estrategias de cara al futuro, teniendo presente el objetivo de haber eliminado del subcontinente la rabia transmitida por perros a más tardar en 2030.

Genetic clustering of recent classical swine fever virus isolates from Karnataka, India revealed the emergence of subtype 2.2 replacing subtype 1.1.

The phylogenetic analysis of 11 CSFV isolates from Karnataka, India obtained during the year 2012-13 was undertaken to obtain the most reliable genetic typing of the CSFV isolates based on E2, NS5B and 5'UTR genomic regions. The study indicated that all the 11 CSFV isolates belonged to subgroup 2.2. The most reliable classification was obtained with sequence data from the NS5B region which separated all the isolates based on the history of outbreak and geographic origin. Analysis of full length E2 amino acid sequences revealed different genetic makeup of Indian 2.2 isolates compared to 2.2 isolates from different countries. The group 2.2 viruses are gradually spreading as confirmed by frequent detection/ isolation of group 2.2 viruses in the recent years and replacing the subgroup 1.1 viruses, which were hitherto predominantly involved in CSF outbreaks in India.

Typing of Staphylococcus aureus isolated from bovine mastitis cases in Australia and India.

OBJECTIVE: To determine the prevalence of the different capsular polysaccharide (CP) and major surface-associated non-CP antigen 336 (SP-336) types among Staphylococcus aureus isolated from bovine mastitis cases in Australia and India. METHODS: A total of 414 strains (154 from Australia, 260 from India) isolated from clinical bovine mastitis were included in the study. Mouse antisera raised against CP types (CP1, CP2, CP5, and CP8) or SP-336 were used in slide agglutination tests and compared with detection of cap1, cap5 and cap8 gene fragments by PCR. RESULTS: Serological studies revealed the presence of CP2, CP5, CP8 and SP-336 in 9.1%, 23.4%, 31.8%, and 5.8% of the Australian versus 0.8%, 46.9%, 13.1% and 0% of the Indian isolates, respectively. By PCR, CP1, CP5 and CP8 accounted for 0%, 26.6% and 32.4% of the Australian versus 3.9%, 85% and 8.1% of the Indian isolates, respectively. CONCLUSIONS: Both PCR and the serological method demonstrated that CP5 and CP8 are the predominant capsular types in Australia, whereas CP5 is the predominant capsular type in India. The study also demonstrated a strong correlation between both methods of typing for CP1, CP5, CP8 and non-typeable S. aureus strains. High-percentage prevalence of non-typeable isolates in both the countries highlights the importance of continued investigations of the identification of unique surface-associated polysaccharide antigens prevalent among S. aureus isolates for the formulation of CP- and SP-based vaccines for bovine mastitis.

Prevalence of classical swine fever in Karnataka, India.

AIM: The present study was conducted to know the current scenario of classical swine fever (CSF) in Bengaluru Urban, Bengaluru Rural, Chikkaballapur, Madikeri, Mandya, Bagalkot, Gadag, Yadgir, Koppal, and Bidar districts of Karnataka with the using of both antigen and antibody ELISA. MATERIALS AND METHODS: We collected 218 sera and 121 blood samples from pigs from 10 different districts of Karnataka. Screening of sera for CSF IgG antibody and whole blood for CSF virus antigen were carried out using the CSF virus (CSFV) antibody and antigen ELISA kits, respectively. RESULTS: The mean seroprevalence was 41% (89/218) and prevalence of CSFV antigen in blood samples was 32% (39/121) for the 10 districts of Karnataka. Seroprevalence of 61%, 29%, 20%, and 21%; and antigen prevalence of 40%, 50%, 13%, and 12% were recorded for Bangalore, Mysore, Belgaum, and Gulbarga divisions of Karnataka, respectively. CONCLUSIONS: The study revealed an alarmingly high prevalence of CSF, both for the antigen (32%) and antibody (41%) in Karnataka. Southern Karnataka has the highest seroprevalence (61% in Bangalore and 29% in Mysore divisions), which confirms the endemicity of the disease in that region. This could be attributed to the intensive pig farming practices in the region as compared to Northern Karnataka (Seroprevalence of 20% in Belgaum and 21% in Gulbarga divisions), where the commercial pig farming is still in infantile stages.

Immunotherapeutics to prevent the replication of Brucella in a treatment failure mouse model.

Outer membrane vesicles (OMVs) from Brucella melitensis and irradiated Brucella neotomae have been shown to be effective vaccines against a B. melitensis challenge in a mouse model. The present study evaluates the efficacy of these two vaccines as immuno-therapeutics in combination with conventional antibiotics against a B. melitensis infection. BALB/c mice chronically infected with B. melitensis were treated for 4 weeks with doxycycline and gentamicin and vaccinated twice during the course of therapy. Antibiotics in sub-therapeutic concentrations were chosen in such a way that the treatment would result in a therapeutic failure in mice. Although no additive effect of vaccines and antibiotics was seen on the clearance of B. melitensis, mice receiving vaccines along with antibiotics exhibited no Brucella replication post-treatment compared to mice treated only with antibiotics. Administration of irradiated B. neotomae along with antibiotics led to higher production of IFN-γ ex vivo by splenocytes upon stimulation with heat inactivated B. melitensis while no such effect was seen by splenocytes from mice vaccinated with OMVs. OMV vaccinated mice developed significantly higher anti-Brucella IgG antibody titers at the end of the treatment compared to the mice that received only antibiotics. The mice that received only vaccines did not show any significant clearance of Brucella from spleens and livers compared to non-treated control mice. This study suggests that incorporating OMVs or irradiated B. neotomae along with conventional antibiotics might be able to improve therapeutic efficacy and control the progression of disease in treatment failure cases.

Differentiation of Staphylococcus aureus and Staphylococcus epidermidis by PCR for the fibrinogen binding protein gene.

Mastitis is one of the most common and burdensome diseases afflicting dairy animals. Among other causes of mastitis, staphylococci are frequently associated with clinical and subclinical mastitis. Although Staphylococcus aureus is the predominant species involved, Staphylococcus epidermidis and other coagulase-negative staphylococci are increasingly being isolated from cases of bovine mastitis. Although Staph. aureus and Staph. epidermidis can be easily differentiated based on their biochemical properties, such phenotypic identification is time consuming and laborious. This study aimed to rapidly identify Staph. aureus and Staph. epidermidis. Accordingly, a multiplex PCR was developed and we found that a single gene encoding the adhesin fibrinogen binding protein could be used to identify and differentiate the two species. Consequently, a multiplex reaction combining a triplex PCR for Staph. aureus and a duplex PCR for Staph. epidermidis was standardized, first using bacterial cultures and then with pasteurized milk spiked with live organisms or DNA extracted from the organisms. The test could specifically detect Staph. aureus and Staph. epidermidis even in the presence of a dozen other organisms. The limit of detection for detecting Staph. aureus and Staph. epidermidis separately was 10 to 100 cfu/mL for simplex PCR and 10(4)cfu/mL for multiplex PCR. Conversely, the limit was 10(6)cfu/mL by multiplex PCR for simultaneous detection of both the organisms when spiked into culture medium or pasteurized milk. Overnight enrichment enhanced the assay sensitivity 100-fold. The assay had a high diagnostic sensitivity and specificity. The application of the test was verified on 602 field isolates of staphylococci that had been characterized earlier by phenotypic methods. Importantly, 25 coagulase-negative isolates were identified as Staph. aureus by the multiplex PCR. The test could be adapted for use in clinical diagnostic laboratories.

Multiplex PCR assay for species identification of bovine mastitis pathogens.

AIM: To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk. METHODS AND RESULTS: A two-tube mPCR assay was developed. The accuracy of the mPCR was evaluated using 56 standard reference strains and 705 strains comprising of E. coli (n = 99), staphylococci (n = 522) and streptococci (n = 84). The threshold of detection of the mPCR assay was 10 fg of genomic DNA and <10(3) CFU ml(-1). A comparative evaluation of mPCR with culture method using 115 milk samples from subclinical mastitis showed mPCR to be more efficacious. Subsequently, the mPCR showed successful detection of target bacteria, when applied directly for the assessment of 36 bulk milk samples. CONCLUSION: The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay will be useful for the detection of mastitis, testing bacteriological safety of milk and for species level differentiation. The assay will be of value in the dairy sector for diagnosis and research. The early and accurate identification of pathogens will enable timely interventions for the treatment and control of bovine mastitis.

Brucellosis in high risk group individuals.

PURPOSE: The purpose of this study was to investigate the seroprevalence of brucellosis among high-risk group individuals, consisting of veterinarians and para-veterinarians, shepherds, butchers and animal owners. METHODS: The present work was carried out at Project Directorate on Animal Disease Monitoring and Surveillance, Bangalore, by using the recently developed indirect enzyme-linked immunosorbent assay (ELISA) for antibodies to Brucella abortus. RESULTS: The results were compared with the conventional serological tests, Rose Bengal plate test and standard tube agglutination test. The result showed that the indirect ELISA was more sensitive than the conventional tests. Of 618 tested, the disease of prevalence was at 41.23% in veterinary inspectors, 30.92% in veterinary assistants, 12.37% in veterinary officers, 6.18% in veterinary supervisors, 6.18% in Group D workers, 2.06% in shepherds and 1.03% in butchers. CONCLUSIONS: This study results highlight the immediate necessity to institute control measures to control Brucellosis.

Epidemiology, zoonotic aspects, vaccination and control/eradication of brucellosis in India.

In India, brucellosis was first recognised in 1942 and is now endemic throughout the country. The disease is reported in cattle, buffalo, sheep, goats, pigs, dogs and humans. B. abortus biotype-1 in cattle and buffaloes and B. melitensis biotype-1 in sheep, goats and man are the predominant infective biotypes. The long-term serological studies have indicated that 5% of cattle and 3% of buffaloes are infected with brucellosis. Economic losses due to brucellosis in livestock are considerable in an agrarian country like India. There is no organised and effective brucellosis control programme in the country. With the indigenous development of serum and milk based ELISA kits, the population survey of the disease has been undertaken on a large scale in several states and plans for the control of the disease through calf-hood vaccination are being worked out. An innovative approach--Bovine Brucellosis Progressive Control Programme (BBPCP) is targeted to overcome the basic problems of ban on cow slaughter, distress sale of animals following the positive serological diagnosis of brucellosis and absence of a disease control strategy. The work plan for the implementation of BBPCP is presented.

Development and field validation of an avidin-biotin enzyme-linked immunosorbent assay kit for bovine brucellosis.

The avidin-biotin enzyme-linked immunosorbent assay (A-B ELISA), for use in surveillance for bovine brucellosis in India was developed and calibrated using the indirect brucellosis ELISA kit of the International Atomic Energy Agency (IAEA) as a reference. The reagents used in the A-B ELISA were as follows: the smooth lipopolysaccharide of Brucella abortus strain 99 (antigen); biotinylated anti-bovine immunoglobulin G (detection antibody); avidin-horseradish peroxidase (conjugate); and O-phenylenediamine dihydrochloride (chromogen). The test results were interpreted using the IAEA software EDI version 2.1.1, which was modified for use in the A-B ELISA. The cut-off percentage positivity value was established using 500 brucellosis-positive and 500 brucellosis-negative serum samples, confirmed with reference to the sample data using the indirect ELISA kit. The overall specificity of A-B ELISA was 98.8% and overall sensitivity was 98.2%. Field validation of the A-B ELISA kit was undertaken in six laboratories in India. Screening of 7,040 cattle and 678 buffalo serum samples from 12 states revealed serological evidence of brucellosis in 8.7% of cattle and 10.2% of buffalo. This kit proved to be robust and performed with a similar sensitivity and specificity to the indirect ELISA. The kit can be supplied at a lower cost than current commercial ELISA kits.

A multiplex approach to molecular detection of Brucella abortus and/or Mycobacterium bovis infection in cattle.

A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortus infection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a "gold standard," was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus (samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortus shedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and of B. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.

A serological survey of bovine brucellosis in India.

A serological survey of brucellosis in cattle and buffalo was performed in 23 States of India. A total of 30,437 bovine samples, comprising 23,284 cattle and 7,153 buffalo (Bubalus bubalis), were screened. The screening initially used the rose bengal plate test: doubtful and positive samples were then titrated in the serum tube agglutination test. The overall prevalence rate of antibodies was 1.9% in cattle and 1.8% in buffalo. In a detailed study of 47 organised farms in the southern State of Karnataka, 207 of 4,995 (4.1%) serum samples from cattle showed titres for brucellosis. This result was in contrast to the low rate of seropositive results reported in cattle owned by individual farmers in Karnataka (0.7% of 2,424 serum samples). In organised farms with a history of abortion, placenta retention and repeat breeding, the prevalence rate was 17%.