Drh. Pudjiatmoko, PhD lahir di Purbalingga,17 April 1959, begitu lulus FKH - IPB pada tahun 1983 ia langsung bekerja menjadi Technical Services pada Produsen Obat Hewan, selanjutnya tahun1985-1986 menjadi Manajer Breeding Farm. Pada tahun 1986-1992 ia menjadi Penguji Vaksin Viral pada Lab Virologi Balai Pengujian Mutu dan Sertifikasi Obat Hewan (BPMSOH) Kementerian Pertanian. Ia memperoleh beasiswa Monbusho program PhD (S3) Applied Veterinary Science di Gifu University, Jepang tahun1993-1997, langsung melanjutkan program Post Doctoral Research tahun 1997-2000 di Gifu University dan Lab Kyoto Biken. Ia ditugasi memimpin Lab Bakteriologi BPMSOH tahun 2002-2005. Ia dikirim ke Jepang sebagai Atase Pertanian KBRI Tokyo tahun 2005-2009, begitu kembali ke Indonesia ia dipercaya sebagai Kepala Balai Besar Pengujian Mutu dan Sertifikasi Obat Hewan (BBPMSOH) tahun 2009-2010, dan ia diamanahi sebagai Direktur Kesehatan Hewan di Ditjen PKH, Kementerian Pertanian tahun 2010 sampai Agustus 2015. Sampai 30 April 2024 sebagai Pejabat Medik Veteriner Ahli Utama.
Peringatan Hari Rabies Sedunia ( World Rabies Day ) tahun 2011 dibuka oleh Gubernur Bali yang diwakili oleh Kepala Dinas Peternakan Provinsi Bali, Ir. I Putu Sumantra,M.App.Sc. didampingi oleh Dirjen Peternakan dan Kesehatan Hewan Kementerian Pertanian RI beserta undangan para Bupati/Wali Kota se Bali atau yang mewakili, Ketua DPRD Provinsi Bali atau yang mewakili, Dirjen Pengendalian Penyakit dan Penyehatan Lingkungan, Kementerian Kesehatan RI, Kepala Badan Karantina Pertanian, Kepala Badan Penanggulangan Bencana Nasional, Ketua Komnas Zoonosis Kemenko Kesra RI, Para Kadisnak dan Kesehatan Hewan dari seluruh Indonesia, Kadis Kesehatan Provinsi Bali, Sumatra Utara, Sumatra Barat, Sulawesi Utara, Sulawesi Tengah, Jawa Barat dan NTT, Perwakilan dari FAO, WHO, UNICEP dan Negara- negara Donor dan para undangan lainya.
Dalam acara ini Kadisnak menyampaikan pertama marilah kita haturkan Pujastuti dan angayu bagia kehadapan Ida Sanghyang Widhi Wasa/Tahan Yang Maha Esa atas Asung Kertha Wara Nugraha-Nya maka kita dapat berkumpul di Artsenter dalam keadaan sehat dan berbahagia mengikuti acara Peringatan Hari Rabies Sedunia ( World Rabies Day ) Tahun 2011 untuk Indonesia. Sumantra menyampaikan sambutan Gubernur Bali pada kesempatan yang berbahagia ini atas nama Pemerintah Provinsi Bali dan masyarakat Bali mengucapkan selamat datang di Bali khususnya kepada seluruh peserta dan undangan dari luar Bali dan penghargaan yang sebesar besarnya kepada semua pihak, khususnya kepada Dirjen Peternakan dan Keswan Kementerian Pertanian RI yang bekerjasama dengan Food and Agriculture Organization ( FAO ) Indonesia telah memilih kembali Provinsi Bali sebagai tempat penyelenggaraan peringatan Hari Rabies Sedunia ( World Rabies Day ) tahun 2011.
Rabies merupakan penyakit zoonosis yang sangat berbahaya karena dapat menyebabkan kematian hingga 100% bagi manusia maupun hewan yang telah terinfeksi. Penyakit Rabies telah menyebabkan kekhawatiran masyarakat di dunia karena telah ditemukan hampir di seluruh dunia. Bahkan saja di negara-negara berkembang, di negara-negara yang sedang berkembang tetapi juga di negara-negara maju, sesuai dengan catatan WHO jumlah orang meninggal akibat rabies melebihi 55.000 orang setiap tahunya, Oleh karena itu maka pengendalian penyakit rabies didunia harus mendapatkan perhatian selutuh negara dan organisasi di dunia.
Kasus rabies di Bali yang terjadi pada tanggal 28 Nopember 2008 merupakan kejadian yang mengejutkan Pemerintah dan masyarakat Bali yang sebelumnya secara historis merupakan daerah yang bebas dari rabies. Penyebarannya yang sangat cepat diseluruh Kabupaten dan Kota se Bali yang disertai dengan kasus gigitan anjing pada masyarakat yang sangat banyak menjadikan suasana yang sangat mengkhawatirkan. Sejak ditemukan penyakit rabies di Bali, hingga saat ini telah menyebabkan kematian sebanyak 132 orang dan telah menyedot dana puluhan milyar rupiah untuk pengendalian dan penanganan kasus gigitan. Sebagai destinasi pariwisata dunia dan pintu gerbang kepariwisataan Indonesia, tentunya kejadian tersebut sangat tidak diharapkan berlangsung lama dan harus segera dilakukan langkah-langkah pengendalian yang strategis agar Provinsi Bali dapat cepat terbebas dari kasus rabies.
Sebagai upaya telah dilaksanakan guna pengendalian pemberantasan dan pembebasan rabies di Bali seperti : Sosialisasi dan penyuluhan kepada masyarakat, vaksinasi, eliminasi, surveilans, pengawasan lalu lintas dan perdagangan hewan penular rabies dan lain sebagainya seperti yang telah diatur dalam Peraturan Pemerintah Daerah Provinsi Bali No. 15 tahun 2009 tentang Penanggulangi Rabies di Provinsi Bali. Evaluasi dan analisa dampak dari seluruh kegiatan dilapangan juga dilakukan guna mendapatkan strategi yang lebih baik dalam pengendalian dan pemberantasannya menuju terwujudnya Program Bali Bebas Kasus Rabies Tahun 2012.
Vaksinasi massal pada hewan penular rabies khususnya anjing salah satu langkah yang telah dilaksanakan. Pelaksanaan vaksinasi massal tahap pertama telah dilaksanakan pada bulan September 2010 hingga Maret 2011 yang bekerjasama dengan lembaga swadaya masyarakat Bali Animal Welirare Asociation ( BAWA ) dengan dukungan pendanaan dari World Society for Protection of Animal ( WSPA ) serta dana APBD Provinsi dan Kabupaten Kota se Bali serta APBN . Pelaksanaan vaksinasi massal rabies tahap II juga telah digelar mulai tanggal 25 Mei hingga 15 September 2011 yang didanai dari APBD Prov.Bali, APBN serta dukungan dari FAO dan WHO.
Pelaksanaan vaksinasi massal tersebut yang disertai dengan kebijakan pemberian VAR terhadap setiap orang yang beresiko akibat tergigit anjing telah menurunkan kasus rabies pada hewan penular rabies maupun kematian pada manusia. Jika pada tahun 2010 ditemukan sebanyak 404 kasus positif rabies pada anjing dari hasil uji laboratorium yang dilakukan oleh Balai Besar Veteriner Denpasar terhadap 3.303 sempel otak anjing yang dikirim dari seluruh Bali. Pada tahun 2011 sampai dengan tanggal 14 September 2011 telah terjadi penurunan yaitu sebanyak 231 ekor anjing yang dicurigai rabies dan sebanyak 67 ekor yang positif dari hasil pengamatan laboratorium. Dari 273 desa di Bali yang pernah ditemukan kasus rabies saat ini sebanyak 174 desa sudah tidak ditemukan lagi kasus selama lebih dari 12 bulan, 33 desa lebih dari 9 bulan dan 23 desa lebih dari 6 bulan.
Kasus kematian pada manusia juga telah terjadi penurunan yang berarti. Selama tahun 2010 jumlah orang meninggal yang dicurigai rabies sebanyak 82 orang dan yang positif secara laboratorium sebanyak 34 kasus, sedangkan selama tahun 2011 sampai dengan hari ini dilaporkan kematian orang yang dicurigai rabies sebanyak 18 orang dan 5 diantaranya yang masih positif secara laboratorium. Rabies di Bali sudah menunjukan penurunan, namun kewaspadaan tetap dilakukan di masyarakat.
Pada tahun 2012 akan dilaksanakan kembali vaksinasi massal rabies tahap III yang akan diikuti dengan langkah pengendalian populasi serta penertiban pemeliharaan dan perdagangan hewan penular rabies khususnya pemeliharaan dan perdagangan anjing. Hal ini sangat perlu dilakukan agar populasi anjing tidak terus berkembang. Populasi anjing di Bali diharapkan bisa dipertahankan sebanyak 150.000 hingga maksimal 200.000 ekor saja dengan pemeliharaan yang baik dan benar. Dengan jumlah dan cara pemeliharaan tersebut maka program vaksinasi akan lebih berhasil yang diikuti dengan berkurangnya kasus gigitan dan tidak lagi terjadi kematian pada manusia sehingga dapat diwujudkanya Bali Bebas Kasus Rabies Tahun 2012. Dengan dukungan dan partisipasi semua pihak baik Nasional maupun Internasional guna mensukseskan cita-cita tersebut sesuai dengan prinsip " One World One Health".
Berdasarkan apa yang telah disampaikan tadi dalam sambutan Gubenur Bali dibacakan oleh Sumantra maka momentum ini saya pandang sangatlah penting bagi kita semua dalam rangka upaya bersama-sama memberantas rabies di dunia, khususnya di Indonesia dan lebih khusus lagi di Provinsi Bali. Dalam acara dan kesempatan ini atas nama Pemerintah Provinsi Bali dan masyarakat Bali juga memberikan penghargaan dan terima kasih yang sebanyak-banyaknya atas partisipasi Yayasan Bali Animal Walfare Asociation ( BAWA ) serta perhatian dan dukungan dari World Society for Protection of Animal (WSPA), FAO, WHO, ACIAR, AusAID. USAID, JSI Deliver serta Pemerintah Pusat dalam pemberantasan rabies di Provinsi Bali. Mudah-mudahan kerja sama ini dapat berjalan dengan baik dan berlangsung terus hingga status Bali Bebas dari Rabies dapat tercapai dan dapat terus dipertahankan.
Daging baik yang belum diolah maupun yang sudah diolah bisa kurang menarik apabila warnanya lemah atau pudar. Sementara masa simpan yang lebih panjang tetap menjadi dambaan para produsen maupun konsumen. Warna daging dapat diperkuat dan masa simpan nya dapat diperpanjang dengan cara mencampurkan senyawa kimia nitrat (NO3) dan atau Nitrit (NO2) pada daging atau produk olahannya.
Yang berperan langsung pada proses perbaikan warna daging adalah nitro-oksigen (NO) yang dihasilkan oleh nitrat dan nitrit. Sedangkan untuk perpanjangan masa simpan yang berperan adalah nitrit. Terjadi reaksi bakteriologis nitrat menghasilkan nitrit lalu berlanjut dengan proses reaksi kimia nitrit menghasilkan NO, masing-masing dengan pengurangan unsur oksigen pada molekulnya.
Pelepasan unsur oksigen pada nitrat dan nitrit merupakan hasil proses reaksi kimia yang berbeda. Reduksi unsur oksigen dari nitrat menjadi nitrit terjadi oleh raksi bakteriologis, sedangkan pengurangan unsur oksigen pada nitrit menjadi nitro-oksigen (NO) merupakan reaksi kimia. Dari nitrat hingga menjadi NO perlu dua langkah. Artinya untuk keperluan proses cepat kehadiran dan aktifitas nitro-oksigen maka penggunaan nitrit lebih tepat karena menghasilkan NO dengan lebih cepat.
Sehingga pada upaya memperbaiki warna produk daging yang sudah dimasak atau direbus yang diutamakan adalah penggunaan nitrit. Hal sama juga berlaku pada upaya perpanjangan masa simpan. Sedangkan untuk daging yang sudah diolah dan difermentasi seperti daging diasini dan sosis yang difermentasi kering, peran nitrat lebih menonjol karena prosesnya lebih lambat dan butuh waktu lebih panjang. Pada daging yang telah diolah dan difermentasi, kultur starter dan bakteri mengubah nitrat menjadi nitrit.
Nitro-oksigen memperbaiki warna melalui reaksi dengan myoglobin dalam daging, suatu reaksi satu arah yang biasanya tidak bisa balik (irreversible). Reaksi tersebut menghasilkan komponen yang stabil, yakni melalui rekasi NO dengan atom besi dalam myoglobin. Proses tersebut yang berlangsung dengan bantuan pemanasan atau lainnya menyebabkan terjadi pengurangan unsur dan denaturasi protein sehingga memberi warna yang lebih baik pada daging. Namun penggunaan nitrat dan nitrit harus berhati-hati karena bila berlebihan bisa menyebabkan oksidasi daging yang menimbulkan pigmen hijau.
Pada efek perpanjangan masa simpan, peran nitrit dan nitrat berada dalam lingkup teknologi penghambatan yang merupakan kombinasi berbagai cara pengawetan. Produk yang dimasak mengutamakan penghambat berupa pemanasan, bahan tambahan pangan (btp), pH dan kandungan air. Produk yang diolah dan difermentasikan mengandalkan kultur starter, pH, kandungan air dan btp. Nitrat dan nitrit merupakan bahan penghambat penting pertumbuhan bakteri seperti Chlostridia sehingga perlu ditambahkan untuk memperpanjang masa simpan.
Daging yang dimasak menghadapi masalah warna dan masa simpan sekaligus. Pemanasan menyebabkan warna menjadi coklat atau abu-abu. Dengan aplikasi nitrat dan nitrit, warna bisa lebih menarik mendekati warna daging normal dan pertumbuhan bakteri bisa terhambat. Daging yang belum diolah atau belum difermentasi lebih rawan terhadap bakteri. Ada beberapa kombinasi penghambat yang telah dikembangkan tanpa penggunaan nitrat dan nitrit. Tetapi dengan penggunaan nitrat dan nitrit warna bisa lebih menarik dan kwlitas produk lebih baik.
Perlu diperhatikan pengaruh yang bisa diilakukan nitrat dan nitrit terhadap daging tipe kering-padat-gelap (dark firm dry / DFD) dan tipe pucat-lembek-basah (pale soft exudative / PSE). Daging tipe DFD memiliki pH tinggi (6,2 - 6,4) sehingga warnanya gelap, daya serap air sangat baik, tetapi stabilitas, warna dan daya simpan tidak bagus. Aplikasi nitrat dan nitrit pada daging DFD yang dimasak memberikan pengaruh positif terhadap warnanya. Pada perlakuan fermentasi, daging tipe DFD tidak bisa digunakan karena pada produk akhir warna tetap akan gelap, pH tetap tinggi dan pertumbuhan bakteri tidak stabil.
Pada daging tipe PSE, warnanya sangat pucat, protein yang terkandung sedikit terdenaturasi dan basah. Bila dimasak, penambahan nitrit akan memberi warna yang lebih baik walaupun tidak menyamai warna daging normal. Secara bakteriologis daging PSE lebih baik dibanding DFD dan menyamai daging normal.
Pada pencegahan oksidasi yang membuat daging menjadi tengik, bahan antioksidan seperti natrium askorbat, vitamin C dan lainnya bisa digunakan. Tetapi dibanding senyawa-senyawa antioksidan, nitrat dan nitrit memiliki kemampuan lebih kuat untuk mencegah ketengikan akibat oksidasi.
Sumber : Sinar Tani Edisi 29 Juni –m 5 Juli 2011 no. 3412 Tahun XLI hal 20.
Pada Sidang Umum Tahunan ke 79 Organisasi Kesehatan Dunia (OIE) di Paris 22-27 Mei 2011 telah dicatat bahwa dunia telah terbebas dari penyakit hewan Rinderpest, dan ancaman sejumlah penyakit utama lainnya pada hewan sudah semakin berkurang. Peserta sidang berjumlah 600 orang delegasi bersemangat membahas cara-cara yang lebih sempurna dan efisien dalam pengendalian berbagai penyakit.
Sidang membahas 118 jenis penyakit hewan di permukaan bumi. Di antara yang paling menyedot perhatian peserta adalah yang terkait dengan Penyakit Mulut dan Kuku (PMK), flu burung, rabies, dan penyakit hewan akuatik.
Tiga penyakit utama yang dievaluasi pada persidangan tersebut adalah penyakit sapi gila (Bovine Spongiform encephalopathy/BSE), PMK, serta Contagious bovine pleuropneumoniae (CBPP).
Tentang penyakit BSE disepakati bahwa Denmark dan Panama yang selama ini dinilai stutus risiko BSE terkendali(controlled risk) kini telah dinyatakan dalam status risiko yang bisa diabaikan (negligible risk).
Tujuh negara yang baru diberikan pengakuan sudah bebas penyakit PMK dengan atau tanpa vaksinasi di seluruh wilayah negara-negara tersebut adalah Jepang, Bostwana, Pilipina, Arhgentina, Bolivia, Brasil, dan Paraguay.
Sedangkan pada saat ini negara yang baru dinyatakan bebas penyakit CBPP adalah China.
Hal lain yang dibahas dalam Sidang Umum OIE 2011 itu termasuk penyempurnaan berbagai aturan dan standar internasional kesehatan dan kesejahteraan hewan termasuk hewan liar. Pembahasan khusus lain termasuk penyakit lebah, kesejahteraan hewan pada produksi ayam potong, dan penyakit rabies pada anjing.
Para delegasi juga memberikan pandangan dan masukan untuk implementasi strategi global pengendalian PMK yang akan diluncurkan dalam waktu dekat.
Sidang menyatakan dukungan terhadap pengembangan kapasitas, dan Iptek OIE. Di antaranya program kerjasama laboratorium Utara-Selatan dan Selatan-Sealatan yang mencakup 38 Laboratorium. Sidang menyepakati akreditasi tiga Pusat Kerjasama (Collaborating Centres) baru dan 11 laboratorium rujukan baru sehingga sekarang terdapat 263 Pusat Ilmiah dalam jaringan global OIE.
Anthrax is an acutedisease caused by the bacterium Bacillus anthracis. Most forms of the disease are lethal, and it affects both humans and other animals. There are effective vaccines against anthrax, and some forms of the disease respond well to antibiotic treatment.
Like many other members of the genusBacillus, Bacillus anthracis can form dormant endospores (often referred to as "spores" for short, but not to be confused with fungal spores) that are able to survive in harsh conditions for decades or even centuries.[1] Such spores can be found on all continents, even Antarctica.[2] When spores are inhaled, ingested, or come into contact with a skin lesion on a host they may reactivate and multiply rapidly.
Anthrax commonly infects wild and domesticated herbivorous mammals that ingest or inhale the spores while grazing. Ingestion is thought to be the most common route by which herbivores contract anthrax. Carnivores living in the same environment may become infected by consuming infected animals. Diseased animals can spread anthrax to humans, either by direct contact (e.g., inoculation of infected blood to broken skin) or by consumption of a diseased animal's flesh.
Anthrax spores can be produced in vitro and used as a biological weapon. Anthrax does not spread directly from one infected animal or person to another; it is spread by spores. These spores can be transported by clothing or shoes. The body of an animal that had active anthrax at the time of death can also be a source of anthrax spores.
Overview
Until the twentieth century, anthrax infections killed hundreds and thousands of animals and people each year in Australia, Asia, Africa, North America, and Europe, particularly in the concentration camps during WWII.[3] French scientist Louis Pasteur developed the first effective vaccine for anthrax in 1881.[4][5][6] Thanks to over a century of animal vaccination programs, sterilization of raw animal waste materials and anthrax eradication programs in North America, Australia, New Zealand, Russia, Europe, and parts of Africa and Asia, anthrax infection is now relatively rare in domestic animals, with only a few dozen cases reported every year. Anthrax is especially rare in dogs and cats, as is evidenced by a single reported case in the USA in 2001. [7] Anthrax typically does not cause disease in carnivores and scavengers, even when these animals consume anthrax-infected carcasses. Anthrax outbreaks do occur in some wild animal populations with some regularity.[8] The disease is more common in developing countries without widespread veterinary or human public health programs.
Bacillus anthracis bacterial spores are soil-borne, and, because of their long lifetime, they are still present globally and at animal burial sites of anthrax-killed animals for many decades; spores have been known to have reinfected animals over 70 years after burial sites of anthrax-infected animals were disturbed.[9]
The virulent Ames strain, which was used in the 2001 anthrax attacks in the United States, has received the most news coverage of any anthrax outbreak. The Ames strain contains two virulence plasmids, which separately encode for a three-protein toxin, called anthrax toxin, and a poly-glutamic acid capsule. Nonetheless, the Vollum strain, developed but never used as a biological weapon during the Second World War, is much more dangerous. The Vollum (also incorrectly referred to as Vellum) strain was isolated in 1935 from a cow in Oxfordshire, UK. This is the same strain that was used during the Gruinard bioweapons trials. A variation of Vollum known as "Vollum 1B" was used during the 1960s in the US and UK bioweapon programs. Vollum 1B is widely believed[10] to have been isolated from William A. Boyles, a 46-year-old scientist at the U.S. Army Biological Warfare Laboratories at Camp (later Fort) Detrick (precursor to USAMRIID) who died in 1951 after being accidentally infected with the Vollum strain. The Sterne strain, named after the Trieste-born immunologist Max Sterne, is an attenuated strain used as a vaccine, which contains only the anthrax toxin virulence plasmid and not the poly-glutamic acid capsule expressing plasmid.
Cause
Bacteria
Bacillus anthracis is a rod-shaped, Gram-positive, aerobic bacterium about 1 by 9 micrometers in length. It was shown to cause disease by Robert Koch in 1876.[11] The bacterium normally rests in endospore form in the soil, and can survive for decades in this state. Herbivores are often infected whilst grazing or browsing, especially when eating rough, irritant, or spiky vegetation: the vegetation has been hypothesized to cause wounds within the gastrointestinal tract permitting entry of the bacterial endo-spores into the tissues, though this has not been proven. Once ingested or placed in an open cut, the bacterium begins multiplying inside the animal or human and typically kills the host within a few days or weeks. The endo-spores germinate at the site of entry into the tissues and then spread via the circulation to the lymphatics, where the bacteria multiply.
It is the production of two powerful exo-toxins and lethal toxin by the bacteria that causes death. Veterinarians can often tell a possible anthrax-induced death by its sudden occurrence, and by the dark, non-clotting blood that oozes from the body orifices. Most anthrax bacteria inside the body after death are out-competed and destroyed by anaerobic bacteria within minutes to hours postmortem. However, anthrax vegetative bacteria that escape the body via oozing blood or through the opening of the carcass may form hardy spores. One spore forms per one vegetative bacterium. The triggers for spore formation are not yet known, though oxygen tension and lack of nutrients may play roles. Once formed, these spores are very hard to eradicate.
The infection of herbivores (and occasionally humans) via the inhalational route normally proceeds as follows: Once the spores are inhaled, they are transported through the air passages into the tiny air particles sacs (alveoli) in the lungs. The spores are then picked up by scavenger cells (macrophages) in the lungs and are transported through small vessels (lymphatics) to the lymph nodes in the central chest cavity (mediastinum). Damage caused by the anthrax spores and bacilli to the central chest cavity can cause chest pain and difficulty breathing. Once in the lymph nodes, the spores germinate into active bacilli that multiply and eventually burst the macrophages, releasing many more bacilli into the bloodstream to be transferred to the entire body. Once in the blood stream, these bacilli release three proteins named lethal factor, edema factor, and protective antigen. All three are non-toxic by themselves, but the combination is incredibly lethal to humans.[12] Protective antigen combines with these other two factors to form lethal toxin and edema toxin, respectively. These toxins are the primary agents of tissue destruction, bleeding, and death of the host. If antibiotics are administered too late, even if the antibiotics eradicate the bacteria, some hosts will still die of toxemia. This is because the toxins produced by the bacilli remain in their system at lethal dose levels.
The lethality of the anthrax disease owes itself to the bacterium's two principal virulence factors: (i) the poly-D-glutamic acid capsule, which protects the bacterium from phagocytosis by host neutrophils, and (ii) the tripartite protein toxin, called anthrax toxin. Anthrax toxin is a mixture of three protein components: (i) protective antigen (PA), (ii) edema factor (EF), and (iii) lethal factor (LF). PA plus LF produces lethal toxin, and PA plus EF produces edema toxin. These toxins cause death and tissue swelling (edema), respectively.
In order to enter the cells, the edema and lethal factors use another protein produced by B. anthracis called protective antigen. Protective antigen binds to two surface receptors on the host cell. A cell protease then cleaves PA into two fragments: PA20 and PA63. PA20 dissociates into the extracellular medium, playing no further role in the toxic cycle. PA63 then oligomerizes with six other PA63 fragments forming a heptameric ring-shaped structure named a prepore. Once in this shape, the complex can competitively bind up to three EF or LF forming a resistant complex.[12] Receptor-mediated endocytosis occurs next, providing the newly formed toxic complex access to the interior of the host cell. The acidified environment within the endosome triggers the heptamer to release the LF and/or EF into the cytosol.[13] It is unknown how exactly the complex results in the death of the cell.
Edema factor is a calmodulin-dependent adenylate cyclase. Adenylate cyclase catalyzes the conversion of ATP into cyclic AMP (cAMP) and pyrophosphate. The complexation of adenylate cyclase with calmodulin removes calmodulin from stimulating calcium-triggered signaling, thus inhibiting the immune response.[12] To be specific, LF inactivates neutrophils (a type of phagocytic cell) by the process just described so that they cannot phagocytose bacteria. Throughout history, it was believed that lethal factor caused macrophages to make TNF-alpha and interleukin 1, beta (IL1B). TNF-alpha is a cytokine whose primary role is to regulate immune cells as well as to induce inflammation and apoptosis or programmed cell death. Interleukin 1, beta is another cytokine that also regulates inflammation and apoptosis. The over-production of TNF-alpha and IL1B ultimately leads to septic shock and death. However, recent evidence indicates that anthrax also targets endothelial cells (cells that line serous cavities such as the pericardial cavity, pleural cavity, and the peritoneal cavity, lymph vessels, and blood vessels), causing vascular leakage of fluid and cells, and ultimately hypovolemic shock (low blood volume), and septic shock.
Exposure
Occupational exposure to infected animals or their products (such as skin, wool, and meat) is the usual pathway of exposure for humans. Workers who are exposed to dead animals and animal products are at the highest risk, especially in countries where anthrax is more common. Anthrax in livestock grazing on open range where they mix with wild animals still occasionally occurs in the United States and elsewhere. Many workers who deal with wool and animal hides are routinely exposed to low levels of anthrax spores but most exposures are not sufficient to develop anthrax infections. It is presumed that the body's natural defenses can destroy low levels of exposure. These people usually contract cutaneous anthrax if they catch anything. Throughout history, the most dangerous form of inhalational anthrax was called Woolsorters' disease because it was an occupational hazard for people who sorted wool. Today this form of infection is extremely rare, as almost no infected animals remain. The last fatal case of natural inhalational anthrax in the United States occurred in California in 1976, when a home weaver died after working with infected wool imported from Pakistan. The autopsy was done at UCLA hospital. To minimize the chance of spreading the disease, the deceased was transported to UCLA in a sealed plastic body bag within a sealed metal container.[14]
In November 2008, a drum maker in the United Kingdom who worked with untreated animal skins died from anthrax.[15] Gastrointestinal anthrax is exceedingly rare in the United States, with only one case on record, reported in 1942, according to the Centers for Disease Control and Prevention.[16] In December 2009 an outbreak of anthrax occurred amongst heroin addicts in Glasgow, Scotland, resulting in ten deaths.[17] The source of the anthrax is believed to be dilution of the heroin with bone meal in Afghanistan.[18]
Also during December 2009, The New Hampshire Department of Health and Human Services confirmed a case of gastrointestinal anthrax in an adult female. The CDC investigated the source and the possibility that it was contracted from an African drum recently used by the woman taking part in a drumming circle.[19] The woman apparently inhaled anthrax [in spore form] from the hide of the drum. She became critically ill, but with gastrointestinal anthrax rather than inhaled anthrax, which made her unique in American medical history. The building where the infection took place was cleaned and reopened to the public and the woman recovered. Jodie Dionne-Odom, New Hampshire state epidemiologist, states, "It is a mystery. We really don't know why it happened."[20] Gastrointestinal anthrax is exceedingly rare in the United States, with only one case on record, reported in 1942, according to the Centers for Disease Control and Prevention.[16]
Mode of infection
Anthrax can enter the human body through the intestines (ingestion), lungs (inhalation), or skin (cutaneous) and causes distinct clinical symptoms based on its site of entry. In general, an infected human will be quarantined. However, anthrax does not usually spread from an infected human to a noninfected human. But, if the disease is fatal to the person's body, its mass of anthrax bacilli becomes a potential source of infection to others and special precautions should be used to prevent further contamination. Inhalational anthrax, if left untreated until obvious symptoms occur, may be fatal.
Anthrax can be contracted in laboratory accidents or by handling infected animals or their wool or hides. It has also been used in biological warfare agents and by terrorists to intentionally infect as exemplified by the 2001 anthrax attacks.
Pulmonary
Respiratory infection in humans initially presents with cold or flu-like symptoms for several days, followed by severe (and often fatal) respiratory collapse. Historical mortality was 92%, but, when treated early (seen in the 2001 anthrax attacks), observed mortality was 45%.[21] Distinguishing pulmonary anthrax from more common causes of respiratory illness is essential to avoiding delays in diagnosis and thereby improving outcomes. An algorithm for this purpose has been developed.[22] Illness progressing to the fulminant phase has a 97% mortality regardless of treatment.
A lethal infection is reported to result from inhalation of about 10,000–20,000 spores, though this dose varies among host species.[23] As with all diseases, it is presumed that there is a wide variation to susceptibility with evidence that some people may die from much lower exposures; there is little documented evidence to verify the exact or average number of spores needed for infection. Inhalational anthrax is also known as Woolsorters' or Ragpickers' disease as these professions were more susceptible to the disease due to their exposure to infected animal products. Other practices associated with exposure include the slicing up of animal horns for the manufacture of buttons, the handling of hair bristles used for the manufacturing of brushes, and the handling of animal skins. Whether these animal skins came from animals that died of the disease or from animals that had simply laid on ground that had spores on it is unknown. This mode of infection is used as a bioweapon.
Gastrointestinal
Gastrointestinal infection in humans is most often caused by eating anthrax-infected meat and is characterized by serious gastrointestinal difficulty, vomiting of blood, severe diarrhea, acute inflammation of the intestinal tract, and loss of appetite. Some lesions have been found in the intestines and in the mouth and throat. After the bacterium invades the bowel system, it spreads through the bloodstream throughout the body, making even more toxins on the way. Gastrointestinal infections can be treated but usually result in fatality rates of 25% to 60%, depending upon how soon treatment commences.[24] This form of anthrax is the rarest form. In the United States, there is only one official case reported in 1942 by the CDC.[16]
Cutaneous
Anthrax skin lesion
Cutaneous (on the skin) anthrax infection in humans shows up as a boil-like skin lesion that eventually forms an ulcer with a black center (eschar). The black eschar often shows up as a large, painless necrotic ulcer (beginning as an irritating and itchy skin lesion or blister that is dark and usually concentrated as a black dot, somewhat resembling bread mold) at the site of infection. In general, cutaneous infections form within the site of spore penetration between 2 and 5 days after exposure. Unlike bruises or most other lesions, cutaneous anthrax infections normally do not cause pain.[24]
Cutaneous anthrax is typically caused when bacillus anthracis spores enter through cuts on the skin. This form of Anthrax is found most commonly when humans handle infected animals and/or animal products (e.g., the hide of an animal used to make drums).
Cutaneous anthrax is rarely fatal if treated,[21] because the infection area is limited to the skin, preventing the Lethal Factor, Edema Factor, and Protective Antigen from entering and destroying a vital organ. Without treatment about 20% of cutaneous skin infection cases progress to toxemia and death.
Treatment typically includes antibiotic therapy. Specific guidelines are available for adults, children, pregnant women, and immunocompromised persons. The differential diagnosis includes multiple entities and thus accurate diagnosis is imperative. Clinical examination coupled with culture and cutaneous biopsy can aid in accurate diagnosis.
Diagnosis
Other than Gram Stain of specimens, there are no specific direct identification techniques for identification of Bacillus species in clinical material. These organisms are Gram-positive but with age can be Gram-variable to Gram-negative. A specific feature of Bacillus species that makes it unique from other aerobic microorganisms is its ability to produce spores. Although spores are not always evident on a Gram stain of this organism, the presence of spores confirms that the organism is of the genus Bacillus.
All Bacillus species grow well on 5% Sheep blood agar and other routine culture media. PLET (polymyxin-lysozyme-EDTA-thallous acetate) can be used to isolate B.anthracis from contaminated specimens, and bicarbonate agar is used as an identification method to induce capsule formation.
Bacillus sp. will usually grow within 24 hours of incubation at 35 degrees C, in ambient air (room temperature) or in 5% CO2. If bicarbonate agar is used for identification then the media must be incubated in 5% CO2.
B.anthracis appears as medium-large, gray, flat, irregular with swirling projections, often referred to as "medusa head" appearance, and is non-hemolytic on 5% sheep blood agar. It is non-motile, is susceptible to penicillin and produces a wide zone of lecithinase on egg yolk agar. Confirmatory testing to identify B.anthracis includes gamma bacteriophage testing, indirect hemagglutination and enzyme linked immunosorbent assay to detect antibodies. [25]
Prevention
Vaccines
An anthrax vaccine licensed by the U.S. Food and Drug Administration (FDA) and produced from one non-virulent strain of the anthrax bacterium, is manufactured by BioPort Corporation, subsidiary of Emergent BioSolutions. The trade name is BioThrax, although it is commonly called Anthrax Vaccine Adsorbed (AVA). It was formerly administered in a six-dose primary series at 0, 2, 4 weeks and 6, 12, 18 months, with annual boosters to maintain immunity. On December 11, 2008, the FDA approved the removal of the 2-week dose, resulting in the currently recommended five-dose series.[26]
Unlike NATO countries, the Soviets developed and used a live spore anthrax vaccine, known as the STI vaccine, produced in Tbilisi, Georgia. Its serious side-effects restrict use to healthy adults.[27]
Treatment
Anthrax cannot be spread directly from person to person, but a patient's clothing and body may be contaminated with anthrax spores. Effective decontamination of people can be accomplished by a thorough wash-down with antimicrobial effective soap and water. Waste water should be treated with bleach or other anti-microbial agent. Effective decontamination of articles can be accomplished by boiling contaminated articles in water for 30 minutes or longer. Chlorine bleach is ineffective in destroying spores and vegetative cells on surfaces, though formaldehyde is effective. Burning clothing is very effective in destroying spores. After decontamination, there is no need to immunize, treat or isolate contacts of persons ill with anthrax unless they were also exposed to the same source of infection.
Antibiotics
Early antibiotic treatment of anthrax is essential—delay significantly lessens chances for survival.
In possible cases of inhalation anthrax, early antibiotic prophylaxis treatment is crucial to prevent possible death.
In May 2009, Human Genome Sciences submitted a Biologic License Application (BLA, permission to market) for its new drug, raxibacumab (brand name ABthrax) intended for emergency treatment of inhaled anthrax.[29] If death occurs from anthrax the body should be isolated to prevent possible spread of anthrax germs. Burial does not kill anthrax spores.
In recent years there have been many attempts to develop new drugs against anthrax, but existing drugs are effective if treatment is started soon enough.
Prevention
If a person is suspected as having died from anthrax, every precaution should be taken to avoid skin contact with the potentially contaminated body and fluids exuded through natural body openings. The body should be put in strict quarantine. A blood sample taken in a sealed container and analyzed in an approved laboratory should be used to ascertain if anthrax is the cause of death. Microscopic visualization of the encapsulated bacilli, usually in very large numbers, in a blood smear stained with polychrome methylene blue (McFadyean stain) is fully diagnostic, though culture of the organism is still the gold standard for diagnosis. Full isolation of the body is important to prevent possible contamination of others. Protective, impermeable clothing and equipment such as rubber gloves, rubber apron, and rubber boots with no perforations should be used when handling the body. No skin, especially if it has any wounds or scratches, should be exposed. Disposable personal protective equipment is preferable, but if not available, decontamination can be achieved by autoclaving. Disposable personal protective equipment and filters should be autoclaved, and/or burned and buried. Bacillus anthracis bacillii range from 0.5–5.0 μm in size. Anyone working with anthrax in a suspected or confirmed victim should wear respiratory equipment capable of filtering this size of particle or smaller. The US National Institute for Occupational Safety and Health (NIOSH) and Mine Safety and Health Administration (MSHA) approved high efficiency-respirator, such as a half-face disposable respirator with a high-efficiency particulate air (HEPA) filter, is recommended.[30] All possibly contaminated bedding or clothing should be isolated in double plastic bags and treated as possible bio-hazard waste. The victim should be sealed in an airtight body bag. Dead victims that are opened and not burned provide an ideal source of anthrax spores. Cremating victims is the preferred way of handling body disposal. No embalming or autopsy should be attempted without a fully equipped biohazard laboratory and trained and knowledgeable personnel.
Delays of only a few days may make the disease untreatable and treatment should be started even without symptoms if possible contamination or exposure is suspected. Animals with anthrax often just die without any apparent symptoms. Initial symptoms may resemble a common cold—sore throat, mild fever, muscle aches and malaise. After a few days, the symptoms may progress to severe breathing problems and shock and ultimately death. Death can occur from about two days to a month after exposure with deaths apparently peaking at about 8 days after exposure.[31] Antibiotic-resistant strains of anthrax are known.
Early detection of sources of anthrax infection can allow preventive measures to be taken. In response to the anthrax attacks of October 2001 the United States Postal Service (USPS) installed BioDetection Systems (BDS) in their large scale mail cancellation facilities. BDS response plans were formulated by the USPS in conjunction with local responders including fire, police, hospitals and public health. Employees of these facilities have been educated about anthrax, response actions and prophylactic medication. Because of the time delay inherent in getting final verification that anthrax has been used, prophylactic antibiotic treatment of possibly exposed personnel must be started as soon as possible.
History
Etymology
The name comes from anthrax [άνθραξ], the Greek word for 'coal', because of the black skin lesions developed by victims with a cutaneous anthrax infection.
Discovery
Robert Koch, a German physician and scientist, first identified the bacterium that caused the anthrax disease in 1875.[11][32] His pioneering work in the late nineteenth century was one of the first demonstrations that diseases could be caused by microbes. In a groundbreaking series of experiments, he uncovered the life cycle and means of transmission of anthrax. His experiments not only helped create an understanding of anthrax, but also helped elucidate the role of microbes in causing illness at a time when debates were still held over spontaneous generation versus cell theory. Koch went on to study the mechanisms of other diseases and was awarded the 1905 Nobel Prize in Physiology or Medicine for his discovery of the bacterium causing tuberculosis. Koch is today recognized as one of history's most important biologists and a founder of modern bacteriology.
First vaccination
In May 1881 Louis Pasteur performed a public experiment to demonstrate his concept of vaccination. He prepared two groups of 25 sheep, one goat and several cows. The animals of one group were injected with an anthrax vaccine prepared by Pasteur twice, at an interval of 15 days; the control group was left unvaccinated. Thirty days after the first injection, both groups were injected with a culture of live anthrax bacteria. All the animals in the non-vaccinated group died, while all of the animals in the vaccinated group survived.[33] The human vaccine for anthrax became available in 1954. This was a cell-free vaccine instead of the live-cell Pasteur-style vaccine used for veterinary purposes. An improved cell-free vaccine became available in 1970.[34]
Society and culture
Site cleanup
Anthrax spores can survive for long periods of time in the environment after release. Methods for cleaning anthrax-contaminated sites commonly use oxidizing agents such as peroxides, ethylene oxide, Sandia Foam,[35] chlorine dioxide (used in Hart Senate office building), and liquid bleach products containing sodium hypochlorite. These agents slowly destroy bacterial spores. A bleach solution for treating hard surfaces has been approved by the EPA.[36] Bleach and vinegar must not be combined together directly, as doing so could produce chlorine gas. Rather some water must first be added to the bleach (e.g., two cups water to one cup of bleach), then vinegar (e.g., one cup), and then the rest of the water (e.g., six cups). The pH of the solution should be tested with a paper test strip; and treated surfaces must remain in contact with the bleach solution for 60 minutes (repeated applications will be necessary to keep the surfaces wet).
Chlorine dioxide has emerged as the preferred biocide against anthrax-contaminated sites, having been employed in the treatment of numerous government buildings over the past decade. Its chief drawback is the need for in situ processes to have the reactant on demand.
To speed the process, trace amounts of a non-toxic catalyst composed of iron and tetro-amido macrocyclic ligands are combined with sodium carbonate and bicarbonate and converted into a spray. The spray formula is applied to an infested area and is followed by another spray containing tert-Butyl hydroperoxide.[37]
Using the catalyst method, a complete destruction of all anthrax spores can be achieved in under 30 minutes.[37] A standard catalyst-free spray destroys fewer than half the spores in the same amount of time. They can be heated, exposed to the harshest chemicals, and they do not easily die.[vague]
Cleanups at a Senate office building, several contaminated postal facilities and other U.S. government and private office buildings showed that decontamination is possible, but it is time-consuming and costly. Clearing the Senate office building of anthrax spores cost $27 million, according to the Government Accountability Office. Cleaning the Brentwood postal facility outside Washington cost $130 million and took 26 months. Since then newer and less costly methods have been developed.[38]
Clean up of anthrax-contaminated areas on ranches and in the wild is much more problematic. Carcasses may be burned, though it often takes up to three days to burn a large carcass and this is not feasible in areas with little wood. Carcasses may also be buried, though the burying of large animals deeply enough to prevent resurfacing of spores requires much manpower and expensive tools. Carcasses have been soaked in formaldehyde to kill spores, though this has environmental contamination issues. Block burning of vegetation in large areas enclosing an anthrax outbreak has been tried; this, while environmentally destructive, causes healthy animals to move away from an area with carcasses in search of fresh graze and browse. Some wildlife workers have experimented with covering fresh anthrax carcasses with shadecloth and heavy objects. This prevents some scavengers from opening the carcasses, thus allowing the putrefactive bacteria within the carcass to kill the vegetative B. anthracis cells and preventing sporulation. This method also has drawbacks, as scavengers such as hyenas are capable of infiltrating almost any exclosure. The occurrence of previously dormant anthrax, stirred up from below the ground surface by wind movement in a drought-stricken region with depleted grazing and browsing, may be seen as a form of natural culling and a first step in rehabilitation of the area.
Biological warfare
Anthrax was first tested as a biological warfare agent by Unit 731 of the Japanese Kwantung Army in Manchuria during the 1930s; some of this testing involved intentional infection of prisoners of war, thousands of whom died. Anthrax, designated at the time as Agent N, was also investigated by the allies in the 1940s. Weaponized anthrax was part of the U.S. stockpile prior to 1972, when the United States signed the Biological Weapons Convention.[39]
Anthrax spores can and have been used as a biological warfare weapon. Its first modern incidence occurred when Scandinavian freedom fighters ("the rebel groups") supplied by the German General Staff used anthrax with unknown results against the Imperial Russian Army in Finland in 1916.[40] There is a long history of practical bioweapons research in this area. For example, in 1942 British bioweapons trials[41] severely contaminated Gruinard Island in Scotland with anthrax spores of the Vollum-14578 strain, making it a no-go area until it was decontaminated in 1990.[42][43] The Gruinard trials involved testing the effectiveness of a submunition of an "N-bomb"—a biological weapon. Additionally, five million "cattle cakes" impregnated with anthrax were prepared and stored at Porton Down in "Operation Vegetarian"—an anti-livestock weapon intended for attacks on Germany by the Royal Air Force.[44] The infected cattle cakes were to be dropped on Germany in 1944. However neither the cakes nor the bomb was used; the cattle cakes were incinerated in late 1945.
More recently, the Rhodesian government used anthrax against cattle and humans in the period 1978–1979 during its war with black nationalists.[45]
American military and British Army personnel are routinely vaccinated against anthrax prior to active service in places where biological attacks are considered a threat. The anthrax vaccine, produced by BioPort Corporation, contains non-living bacteria, and is approximately 93% effective in preventing infection.[citation needed]
Weaponized stocks of anthrax in the US were destroyed in 1971–72 after President Nixon ordered the dismantling of US biowarfare programs in 1969 and the destruction of all existing stockpiles of bioweapons.
The Soviet Union created and stored 100 to 200 tons of anthrax spores at Kantubek on Vozrozhdeniya Island. They were abandoned in 1992 and destroyed in 2002.
Despite signing the 1972 agreement to end bioweapon production the government of the Soviet Union had an active bioweapons program that included the production of hundreds of tons of weapons-grade anthrax after this period. On 2 April 1979, some of the over one million people living in Sverdlovsk (now called Ekaterinburg, Russia), about 850 miles east of Moscow, were exposed to an accidental release of anthrax from a biological weapons complex located near there. At least 94 people were infected, of whom at least 68 died. One victim died four days after the release, ten over an eight-day period at the peak of the deaths, and the last six weeks later. Extensive cleanup, vaccinations and medical interventions managed to save about 30 of the victims.[46] Extensive cover-ups and destruction of records by the KGB continued from 1979 until Russian President Boris Yeltsin admitted this anthrax accident in 1992. Jeanne Guillemin reported in 1999 that a combined Russian and United States team investigated the accident in 1992.[46][47][48]
Nearly all of the night shift workers of a ceramics plant directly across the street from the biological facility (compound 19) became infected, and most died. Since most were men, there were suspicions by NATO governments that the Soviet Union had developed a sex-specific weapon.[49] The government blamed the outbreak on the consumption of anthrax-tainted meat and ordered the confiscation of all uninspected meat that entered the city. They also ordered that all stray dogs be shot and that people not have contact with sick animals. There was also a voluntary evacuation and anthrax vaccination program established for people from 18–55.[50]
To support the cover-up story Soviet medical and legal journals published articles about an outbreak in livestock that caused GI anthrax in people having consumed infected meat, and cutaneous anthrax in people having come into contact with the animals. All medical and public health records were confiscated by the KGB.[50] In addition to the medical problems that the outbreak caused, it also prompted Western countries to be more suspicious of a covert Soviet Bioweapons program and to increase their surveillance of suspected sites. In 1986, the US government was allowed to investigate the incident, and concluded that the exposure was from aerosol anthrax from a military weapons facility.[51] In 1992, President Yeltsin admitted that he was "absolutely certain" that "rumors" about the Soviet Union violating the 1972 Bioweapons Treaty were true. The Soviet Union, like the US and UK, had agreed to submit information to the UN about their bioweapons programs but omitted known facilities and never acknowledged their weapons program.[49]
Anthrax bioterrorism
In theory, anthrax spores can be cultivated with minimal special equipment and a first-year collegiate microbiological education, but in practice the procedure is difficult and dangerous. To make large amounts of an aerosol form of anthrax suitable for biological warfare requires extensive practical knowledge, training, and highly advanced equipment.[citation needed]
Concentrated anthrax spores were used for bioterrorism in the 2001 anthrax attacks in the United States, delivered by mailing postal letters containing the spores.[52] The letters were sent to several news media offices as well as to two Democratic senators: Tom Daschle of South Dakota and Patrick Leahy of Vermont. As a result, 22 were infected and five died.[12] Only a few grams of material were used in these attacks and in August 2008 the US Department of Justice announced they believed that Dr. Bruce Ivins, a senior biodefense researcher employed by the United States government, was responsible.[53] These events also spawned many anthrax hoaxes.
Due to these events, the U.S. Postal Service installed biohazard detection systems at its major distribution centers to actively scan for anthrax being transported through the mail.[54]
A scientific experiment performed by a high school student, later published in The Journal of Medical Toxicology, suggested that a domestic electric iron at its hottest setting (at least 400 °F (204 °C)) used for at least 5 minutes should destroy all anthrax spores in a common postal envelope.[56]
2.^ Hudson JA, Daniel RM, Morgan HW (2006). "Acidophilic and thermophilic Bacillus strains from geothermally heated antarctic soil". FEMS Microbiol Lett60 (3): 279–282.
3.^ Cherkasskiy, B. L. (1999). "A national register of historic and contemporary anthrax foci". Journal of Applied Microbiology87 (2): 192–195. doi:10.1046/j.1365-2672.1999.00868.x. PMID10475946.
8.^ Dragon, D. C.; Elkin, BT; Nishi, JS; Ellsworth, TR (1999). "A review of anthrax in Canada and implications for research on the disease in northern bison". Journal of Applied Microbiology87 (2): 208. doi:10.1046/j.1365-2672.1999.00872.x. PMID10475950.
14.^ Suffin, S. C.; Carnes, W. H.; Kaufmann, A. F. (September 1978). "Inhalation anthrax in a home craftsman". Human Pathology9 (5): 594–7. doi:10.1016/S0046-8177(78)80140-3. PMID101438.
21.^ ab Bravata DM, Holty JE, Liu H, McDonald KM, Olshen RA, Owens DK (February 2006). "Systematic review: a century of inhalational anthrax cases from 1900 to 2005". Ann Intern Med144 (4): 270–80. PMID16490913.
40.^ Bisher, Jamie, "During World War I, Terrorists Schemed to Use Anthrax in the Cause of Finnish Independence," Military History, August 2003, pp. 17–22. Anthrax Sabotage in Finland. Archived 25 October 2009.
49.^ ab Alibek, K. (1999). Biohazard. New York: Delta Publishing. ISBN0385334966.
50.^ ab Meselson, M.; et al., J; Hugh-Jones, M; Langmuir, A; Popova, I; Shelokov, A; Yampolskaya, O (1994). "The Sverdlovsk anthrax outbreak of 1979". Science266 (5188): 1202–1208. doi:10.1126/science.7973702. PMID7973702.
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