To the Editor: In India, buffalopox virus (BPXV), a variant of vaccinia virus, is associated with severe disease outbreaks among buffaloes (1,2), cattle (3), and humans in contact with these animals (1,4). Most human BPXV infections occur in animal attendants and milkers (1,4). A similar type of vaccinia virus infection has also been reported from rural areas in Brazil (5). We report a case of laboratory-acquired infection with BPXV in a researcher in India. Clinical signs, symptoms, diagnosis, and management of this case highlight the need for observance and enforcement of strict biosafety measures within the laboratory.
A 28-year-old man (researcher) who was freeze-drying BPXV isolates in a laboratory in Hisar, India sustained a cut on his right palm through nitrile gloves by accidental piercing of shrapnel from a broken ampule. The virus being freeze-dried (105.5 50% tissue culture infectious doses/mL) was isolated from a buffalo in Jalgaon, India, in 2010. The injured site on the palm was immediately cleaned with 70% ethanol and treated with povidone–iodine solution. No untoward reaction was observed ≤2 days postinjury. Erythema appeared at the injury site on postinjury day 3. Subsequently, a small vesicle developed on postinjury day 5 (Figure, panel A). This vesicle progressed into a pustule with a central area of necrosis by postinjury day 7 (Figure, panel B). On postinjury day 9, symptoms worsened (onset of high fever and general malaise and pain at the affected site), and the researcher sought medical care.
Figure
Progression of lesion on palm of researcher caused by buffalopox virus infection, India. A) Small vesicle on postinjury day 5. B) Pustule with a central area of necrosis on postinjury day 7. C) Pustule with edema on postinjury day 9. D) Increase in edema ...
Physical examination showed high fever (104°F), unilateral axillary lymphadenopathy, and edema of the palm (Figure, panel C). Amoxicillin (500 mg, 2×/d), cephalexin (500 mg, 2×/d), and analgesic/antipyretic (paracetamol, 500 mg, 2×/d) were prescribed to control secondary complications caused by bacterial infection and pain.
The next day, the entire palm became cyanotic and edema increased (Figure, panel D). The researcher was then referred to a specialty hospital where the lesion was surgically excised on postinjury day 11 (Figure, panel E) under axial block anesthesia. Blood, necrotic tissue, pustular material, and swab specimens were obtained for laboratory examination. Postsurgery treatment included cleaning of the surgical site on alternate days and oral medication (amoxicillin/clavulanic acid, 625 mg; ibuprofen, 400 mg; paracetamol, 325 mg; and rabeprazole, 20 mg) for 5 days.
On postinjury day 19, the surgeon advised the patient to take cefuroxime (500 mg/d for 5 days) and use a topical ointment containing mupirocin to prevent a delay in healing (Figure, panel F). The lesion healed slowly, and by postinjury day 30, thickening and blackening of the skin was observed (Figure, panel G) that extended to a wider area by postinjury day 38, and the skin started to peel off by postinjury day 50. The entire skin of the palm sloughed off with complete healing by postinjury day 85, leaving a 20-mm blackened eschar over the area (Figure, panel H).
Clinical samples were subjected to laboratory examination. Virus was isolated from tissue samples in a Vero cell line during the first passage. BPXV infection was confirmed by PCR amplification of orthopoxvirus-specific A type inclusion gene (552 bp) and a BPXV-specific C18L gene (368 bp) from tissue material and the laboratory-isolated virus (BPXV/Human/Lab/11), according to procedures described by Singh et al. (6). Sequences of these 2 genes were submitted to GenBank under accession nos. {type:entrez-nucleotide,attrs:{text:JN653284.1,term_id:346682495,term_text:JN653284.1}}JN653284.1 and {type:entrez-nucleotide,attrs:{text:JN653278.1,term_id:346682489,term_text:JN653278.1}}JN653278.1, respectively. Phylogenetic analysis showed 95%–100% nt similarity of the laboratory isolate (BPXV/Human/Lab/11) with other BPXVs from India (Technical Appendix Figure). Antibodies against BPXV were detected in patient serum samples by using an indirect immunoperoxidase test and a 50% plaque-reduction neutralization test according to methods reported by Bera et al. (7). Serum of the infected patient showed 50% plaque-reduction neutralization test titers of 256 and 512 on postinjury days 11 and 28, respectively. These findings confirmed BPXV infection because the patient had not been vaccinated against smallpox.
Freeze-drying of the glass ampule to −80°C caused a hairline crack in the glass. The ampule broke while being introduced into the freeze-drying manifold and pierced the palm of the researcher. As a follow-up measure, the freeze drying procedure was reviewed and the pre-freezing temperature was reduced to −60°C. Measures were also taken to ensure use of better-quality ampules. Surgical and contact material associated with the lesion was placed in biohazard bags for autoclaving before disposal. In addition, laboratory and hospital staff was apprised of the risk associated with BPXV transmission.
Reporting of laboratory-acquired infections is crucial because infections could also spread to other personnel. Strict biosafety practices and laboratory guidelines are useful in minimizing laboratory-acquired infections. Guidelines, no matter how stringent, are not sufficient on their own. Laboratory-acquired infections occur because humans or machines are not infallible. Thus, laboratories should have emergency procedures in place to deal with such situations.
Technical Appendix:
Phylogenetic trees of A-type inclusion and C18L genes buffalopox virus and related viruses.
Click here to view.(300K, pdf)
Water buffalo are of enormous economic significance in the dairy economy of north India, however, they are highly susceptible to bacterial respiratory diseases including haemorrhagic septicaemia (HS) caused by Pasteurella multocida. An outbreak of HS caused by Pasteurella multocida sub spp. multocida occurred in 3 buffalo calves of a discrete population kept in an organized farm. The peracute episode of disease resulted in early death and samples from animals and post-mortem samples yielded typical bi-polar staining Gram-negative bacilli. The isolations were made from nasal swabs and intestinal contents. T hree isolates of Pasteurella multocida was isolated in pure culture from 2 calves, and the diagnosis was based on clinical signs, biochemical and phenotypic characterization and PCR confirmation performed on the cultures. The calves were diagnosed with HS due to infection with P. multocida capsular type B:2 strain. The cultures were subjected to antimicrobial sensitivity profile, and were found to be generally susceptible except showing resistance to flouroquinolones. The importance of diagnosis, reporting and isolation, preservation and genetic characterization of isolates with geographical information is discussed.
A role for bacteriophage therapy was envisaged early last century; however, due to discovery of the antimicrobials, it fell out of research interest. Currently, bacteriophages are resurfacing as an alternative to antimicrobials in order to overcome the increasing incidence of antimicrobial resistance. Here, we report isolation of bacteriophages against Escherichia coli, Shigella spp., Aeromonas hydrophila and Citrobacter sedlakii isolates of equine origin. Phages were isolated from equine farm soil and sewage samples. For enrichment, sample aliquot was incubated overnight with host bacteria at 37 C with vigorous shaking. The crude lysate obtained was centrifuged and filtered and the presence of any phage in the suspension was detected by agar overlay technique. Appropriate dilutions of enriched samples were plated to obtain individual plaques and themost dominant plaquewas transferred into SM buffer, serially diluted and plated for plaque re-isolation three times to ensure purity, followed by large scale preparation of phage stocks. Any host nucleic acids was degraded using pancreatic DNaseI and RNase and bacteriophage particles were precipitated using PEG8000. Phage titre was determined by plaque assay and phage concentrates were accessioned in the Veterinary Type Culture Collection (VTCC) repository. The phage concentrates were visualized by transmission electron microscopy (TEM). The temperature stability of bacteriophages was checked after incubating phage concentrates over the range of 4 C 80 C temperature for one hour. A clear single plaque was obtained on nutrient agar against Shigella spp. and after purification and concentration, its analysis by electron microscopy revealed presence of multiple phages belonging to families Myoviridae, Siphoviridae and Podoviridae. However in case of Citrobacter sedlakii, a Siphoviridae phage (VTCCBPA61) with dimensions: 60 nm x 650 nm was observed. Against a pathogenic A. hydrophila isolate of equine origin (expressing aerolysin gene), a Myoviridae phage (VTCCBPA6) was isolated with dimensions: 62 nm x 138 nm. Against E. coli of equine origin, a Myoviridae phage (VTCCBPA9) of dimensions: 86 nm x 100 nm was obtained. Bacteriophage VTCCPBA61 against C. sedlakiiwas found to completely lose its biological activity at 65 C in vitro however the group of phages against Shigella spp. were found to be stable upto temperature as high as 80 C. Thus we demonstrated the basic biological characteristics of phages, and some novel ones (such as against A. hyrophila and C. sedlakii) bacterial isolates of equine originwhich have never been reported till now. These lytic phages could find potential in phage therapy, as biocides, in biosensors and in phage ligand technology and are being explored by us further to depict their therapeutic value in small animal model. As more studies are reporting safety, tolerance and efficacy of phage therapy in humans and animals, their use in phage therapy has a promising future as an emerging alternative to chemical agents. Posters 006 Microbiological investigation into the cause of Equine Grass Sickness
Glanders, a bacterial disease of equines caused by Burkholderia mallei, is a fatal infectious disease of equines and has zoonotic significance. The disease has been eradicated from many countries by statutory testing, elimination of infected animals and import restrictions. However, it is still endemic in parts of Africa, Asia, the Middle East and Central and South America. In India, major glanders outbreaks were reported from different parts of the country between 1976 and 1982. Later, sporadic cases of the disease were reported in 1988, 1990 and 1998. The country remained free of glanders for about eight years until the recent outbreaks occurred in eight States from 2006 to 2007. Recurrent episodes have occurred in Himachal Pradesh and Uttar Pradesh, whereas fresh outbreaks occurred in Chhattisgarh from 2009 to 2010. A total of 164 equines were declared positive; a majority of the positive cases (n=77) were from Uttar Pradesh, followed by Maharashtra (n=23), Uttarakhand (n=21) and Andhra Pradesh (n=16). Under the provision of Prevention and Control of Infectious and Contagious Disease in Animals Act, 2009, all the infected animals were euthanised and bio-security measures were implemented to curb the further spread of the disease.
An outbreak of contagious ecthyma was investigated in an organized goatfarm in village Tyod in Jaipur district of Rajasthan. The outbreak occurred during the first week of June, 2012 in Beetal goats purchased from Punjab. Within a week, all the goats (24 females and 1 male) were affected with skin lesions accompanied by anorexia and fever. On the basis of clinical symptoms and the extent of gross lesions, animals were categorized into three groups viz., phase I, IIand III. Of the 25 Beetal goats, four females and one male showed initial lesions (Phase I) such as papules and pustules at the oral commissures and muzzle. Twelve goats showed moderate lesions (Phase II) of ecthyma. Eight goats were highly emaciated and exhibited anorexia, pyrexiaand severe lesions (Phase III) at the oral commissures, muzzle and nostrils as well as haemorrhagic ulcers in buccal mucosa. With the passage of time, the infection spread to other goat flocks of Jakhrana and non-descript goats in which two females of each breed exhibited phase II lesions. Of the 20 kids, only one male kid of Beetal goat was found affected with anorexia, fever and phase I lesions of the disease. The presence of contagious ecthyma in the affected goats was confirmed by specific polymerase chain reaction. Jamunapari, Sirohi, Totapari goats and bucks kept for commercial purposes at the premises did not reveal any clinical signs of the disease. Categorization of the affected animals made it easy to offer an effective treatment as well as prevention of spread of infection in non-affected goats.
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