Bacterial Diseases: Anthrax PDF

PART-1 BACTERIAL DISEASES 1  ANTHRAX Synonyms: Malignant pustule, malignant carbuncle, charbon, hematic anthrax, bacterial anthrax, splenic fever, woolsorters’ disease. Introduction: Anthrax also called Malignant pustule, Malignant...

PART-1 BACTERIAL DISEASES 1  ANTHRAX Synonyms: Malignant pustule, malignant carbuncle, charbon, hematic anthrax, bacterial anthrax, splenic fever, woolsorters’ disease. Introduction: Anthrax also called Malignant pustule, Malignant oedema, Woolsorter's disease, or Ragpicker's disease, is an acute infectious disease of animals caused by Bacillus anthracis, a gram-positive,spore-forming bacillus. Spores of B.anthracis can persist in the environment for many years in some types of soil and enter the body through skin abrasions, inhalation or ingestion and multiply to produce exotoxins. Anthrax is primarily a disease of herbivorous animals that occasionally affects human. In nature, B. anthracis occurs in a virulent form—the pathogenic agent of anthrax— and in an avirulent form. Virulence is determined by a capsule that inhibits phagocytosis and an exotoxin, both of which are plasmid mediated. In turn, the toxin consists of three protein factors: the protective antigen, the lethal factor, and the edema factor. None of these factors is toxic by itself. When injected intravenously at the same time, the protective antigen and the lethal factor are lethal in some animal species. The combination of the protective agent and the edema factor produces edema when injected subcutaneously (Little and Knudson, 1986) (Thomas G Hull. Disease Transmitted from Animals to Man, 5th ed, 1963: 82-125.) Epidemiology: Distributed worldwide, with areas of enzootic and sporadic occurrence. Many regions in India are still enzootic for animal anthrax and sporadic cases of human anthrax have been reported, especially from South India (Tamil Nadu, Puducherry, Anthra Pradesh and Karnataka).  Andhra Pradesh: Chittoor, Cuddapah, Gundur and Prakasam.  Karnataka: Mysore, Midnapore and Kolar. 2 Current Indian Scenario: Anthrax is enzootic in southern India but is less frequent to absent in the northern Indian states. In the past years the anthrax cases have been reported from Andhra Pradesh, Jammu and Kashmir, Tamil Nadu, Orissa and Karnataka. Outbreaks of Anthrax have been reported from Mysore 1999, Orissa 2004, 2005, West Bengal 2000. Transmission: Animals usually become infected by ingestion of contaminated soil or feeds. Infected animals shed the bacilli during terminal hemorrhage, or if the blood of the dead animal is spilled accidentally. On exposure to the air, the vegetative forms sporulate. These spores are markedly resistant to many disinfectants and adverse environmental conditions and remain viable in the contaminated soil for many years. Dried or otherwise processed skins of infected animals may also harbor the spores for years. Thus, the spore forms are predominant in the environment and it is very largely through the uptake of spores that anthrax is contracted. Cutaneous anthrax is the most common anthrax infection. Transmission occurs after exposure to infected animals and contaminated animal products such as hair, hides, wool, bones, or skin. Inhalation anthrax results from inhalation of spores in particles less than 5 micrometer in diameter that may reach the terminal alveoli of the lungs. Aerosols of such particles may be created by the agitation of the hair or wool in the industry settings. Intestinal and oropharyngeal anthrax results from ingestion of contaminated meat. There is no evidence that milk from infected animal transmits anthrax. The disease spreads among omnivores and carnivores through contaminated meat, bonemeal and other feeds and among wild life from feeding on anthrax carcasses. Vultures have been reported to spread the organism from one area to another. Accidental infection may occur among laboratory workers. Direct person to person spread of anthrax is extremely rare. However, precautions should be taken with drainage and secretions of patients to prevent cutaneous anthrax. Second attacks can occur, but are rare. The disease mostly affects adults, especially males. It is due to high exposure rate among these groups. Anthrax is a seasonal disease. Climate probably acts directly or indirectly by influencing the way in which the animal comes into contact with the spores, for example, grazing closer to the soil in dry periods when grass is short and sparse.( Davaine C. Compt rend Acad sc. 1863; 57:386-87.) 3 Anthrax. Transmission cycle. Clinical manifestations: In animals Important clinical manifestations in animals: In ruminants, sudden death, bleeding from orifices, subcutaneous haemorrhage, without priorsymptoms or following a brief period of fever and disorientation should lead to suspicion of anthrax. In equines and some wild herbivores, some transient symptoms such as fever, restlessness, dyspnoea or agitation may be apparent. In pigs, carnivores and primates, local oedema and swelling of face and neck or of lymph nodes,particularly mandibular and pharyngeal and/or mesenteric may be present. The incubation period in the susceptible herbivore ranges from about 36 to 72 hours. The first signs of an anthrax outbreak are one or more sudden deaths in the affected livestock. Other signs include: going off feed, or producing less milk than usual. 4 During the systemic phase, the animals become distressed, appear to have difficult breathing and cease eating and drinking. Swellings in the submandibular fossa may be apparent, and temperature may rise. If the animal fails to respond to the treatment, it lapses into coma followed by death from shock. (PAHO, © 2001.) In humans: Anthrax infection occurs in three forms: cutaneous, inhalation, and gastrointestinal depending on the mode of transmission. Symptoms of disease vary depending on how the disease was contracted, but symptoms usually occur within seven days. Cutaneous anthrax: Most anthrax infections occur when the bacterium enters a cut or abrasion on the skin, such as when handling contaminated wool, hides, leather or hair products of infected animals. The incubation period for cutaneous anthrax is 1-7 days. Skin infection begins as a painless, pruritic papule that resembles an insect bite but within 1-2 days develops into a vesicle (usually 1-3 cm in diameter) and then a painless ulcer with a characteristic black necrotic (dying) area in the center. Systemic symptoms are mild and may include malaise and low-grade fever. There may be regional lymphangitis and lymphadenopathy. Occasionally more severe form of cutaneous anthrax may occur with extensive local oedema, induration and toxemia. The infection can also spread to the bloodstream with overwhelming septicemia. About 20% of untreated cases of cutaneous anthrax will result in death. Deaths are infrequent with appropriate antimicrobial therapy. Inhalation anthrax: Initial symptoms may resemble a common cold. After several days, the symptoms may progress to severe breathing problems and shock. Mediastinal widening is seen in the X-Ray chest. Diagnosis is difficult but inhalation anthrax should be suspected if there is a history of exposure to an aerosol that contains B.anthracis. Inhalation anthrax usually results in death in 1-2 days after onset of the acute symptoms. Intestinal anthrax: The intestinal disease form of anthrax may follow the consumption of contaminated meat and is characterized by an acute inflammation of the intestinal tract. There are two clinical forms of intestinal anthrax. Intestinal anthrax: Symptoms include nausea, vomiting, fever, abdominal pain, haematemesis, bloody diarrhoea and massive ascites. Unless treatment starts early toxaemia and shock develop resulting in death. 5  Oropharyngeal anthrax: Main clinical features are sore throat, dysphagia, fever, lymphadenopathy in the neck and toxaemia. Even with treatment mortality is high, about 50%. Gastro-intestinal anthrax is difficult to diagnose as the symptoms are non-specific. However, history of ingesting meat of a sick animal and cases of cutaneous anthrax in the area may support diagnosis. Intestinal anthrax results in death in 25% to 60% of cases. Meningitis may complicate any of the three primary forms. It resembles meningitis due to other causes although it is frequently haemorrhagic. (Thomas G Hull. Disease Transmitted from Animals to Man, 5th ed, 1963: 82-125.) Diagnosis: 1.Laboratory Diagnosis Laboratory diagnosis for anthrax should be attempted only by laboratory well trained to do so. High index of suspicion of the disease is important. Collection and transportation should be carried out under strict aseptic condition. Human Anthrax In human anthrax ,the bacillus is usually demonstrated in material from a malignant pustule (the best specimen is fluid from an unbroken vesicle at the edge of the lesion) (a) Cutaneous Anthrax In early stage vesicular exudate from the lesions by sterile swab can be collected In later stage material to be taken from underneath of eschar after lifting up of eschar with sterile forcep. The swab should be put in Carry-Blair transport medium and with another swab smear on microscopic slide may be prepared and heat fixed. (b) Intestinal Anthrax If patient is not severely ill, a faecal specimen can be collected If patient is severely ill ascitic fluid (peritoneal fluid) can be collected. (c) Pulmonary Anthrax If patient is not severely ill, sputum can be collected. If patient is severely ill, bronchial levage can be collected. Animal Anthrax 6 Anthrax should be considered as possible cause of death in herbivore's that has died suddenly with haemorrhages for nose, mouth or anal orifice. Samples to be collected (a) Caracass 1 to 2 days old Unclotted blood from nasal, buccal or anal orifice may be collected. If opened; body fluid, spleen and/or unclotted blood may be collected. (b) Old putrefying carcasses Selection of tissues or any blood stained material may be collected. If opened then body fluid, spleen may be collected. (c) If buried Soil or other material from burial site may be collected. 2.Isolation from clinical specimens: a. Sputum specimens Inoculate 3 routine media for sputum specimens (i.e. SBA, MacConkey agar, broth enrichment). b. Blood specimens b.1. Routine blood culture methods are sufficient. b.2. There may be enough organisms in the blood to see them on direct smears by Gram stain. B.anthracis appears as short chains of 2-4 cells which are encapsulated as evidenced by clear zones around the bacilli. The presence of large encapsulated gram-positive rods in the blood is strongly presumptive for B. anthracis Identification. b.3. If blood culture bottle is positive, perform a Gram stain directly and observe for encapsulated rods. These blood cultures should also be subcultured to SBA and MacConkey agar plates. c. Swab specimens c.1. Use one swab to Inoculate 3 standard media for surface wounds (e.g., SBA, MacConkey agar, or broth enrichment). c.2. Prepare a smear for Gram staining with the second swab. d. Stool specimens d.l. Routine stool culture methods are sufficient (e.g., SBA, MacConkey agar, or PEA plates). d.2. Do not use CVA or hectone agar plates. e. CSF specimens e.1. If a clinical centrifuge with appropriate bio containment tube holders is available, centrifuge the CSF specimen at 1500 X g for 15 minutes. e.2. Collect the sediment and prepare a smear for Gram staining. 7 e.3. Inoculate the remainder of the sediment onto SBA and broth enrichment media (tryptic soybroth or thioglycollate). 3. Incubation and examination of cultures a. Cultures should be incubated at 35-37° C under ambient conditions. b. Cultures should be examined within 18-24 h of incubation. Growth of B. anthracis may be observed as early as 8 h after inoculation. 4.Confirmatory tests for Bacillus anthracis A. Animal pathogenicity  5-10 mice injected I/P with suspension of culture material. Observe for mortality.  100% mortality within 24 hours in case of B.anthracis.  If there is no mortality, observe for 10 days.  Material (spleen) from dead mice processed for detection of Bacillus anthracis. B. Molecular methods  Direct PCR.  Study of the genetic make up to detect any engineered strains. Pearl – string test: When Bacillus anthracis is grown for 2-3 hours on solid media containing 0.05 to 0.5 IU penicillin / ml, due to impairment of cell wall, the bacilli become spherical in appearance resembling a string of pearls. This test is an adjunct to the rapid recognition of anthrax bacilli. Ascoli Test: In 1911, Ascoli published a procedure for the detection of thermostable anthraxantigen in animal tissue being used for by-products. This uses antiserum raised in rabbits to produce a precipitin reaction. The test lacks high specificity, in that the thermostable antigens of B. anthracis are shared by other Bacillus spp., and is dependent on the probability that only B. anthracis would proliferate throughout the animal and deposit sufficient antigen to give a positive reaction.Antiserum is prepared in rabbits by the subcutaneous inoculation of Sterne anthrax vaccine on days 1 and 14. On days 28 and 35, the rabbits receive 0.5 ml of a mixture of several strains of virulent B. anthracis not exceeding 105 colony-forming units (CFU)/ml suspended in saline. Alternatively, the live virulent bacteria can be inactivated by prolonged suspension in 0.2% formalised saline, but the antigen mass needs to be 8 increased to 108–109 CFU/ml. The suspension should be checked for inactivation of the B.anthracis before animal inoculation by culture of 0.1 ml into 100 ml of nutrient broth containing 0.1% histidine and, after incubation at 37°C for 7 days, subculture on to blood or nutrient agar. The dose regimen for the formalised suspension after initial vaccination on days 1 and 14 is increasing doses of 0.1, 0.5, 1, and 2 ml given intravenously at intervals of 4–5 days. Following either procedure, a test bleed at 10 days after the last injection should determine whether additional 2 ml doses should be administered to boost the precipitin titre. Procedure: To perform the Ascoli test:  Put approximately 2 g of sample in 5 ml of saline containing 1/100 final concentration of acetic acid.  Boil for 5 minutes.3. The resultant solution is cooled.  Filtered through filter paper.  A few drops of rabbit antiserum (see preparation below) are placed in a small test tube.  The filtrate from the previous step is gently layered over the top of the antiserum Result: A positive test is the formation of a visible precipitin band in under 15minutes. Positive and negative control specimen suspensions should be included. Prevention and control: The problem of anthrax continues because of the following: The custom of butchering and eating roasted meat from sudden death animals and utilizing their hair, hides, bones etc. Lack of cooperation over reporting sudden deaths Long delays in diagnosis due to poor communication and inadequate local laboratory facilities. Failure to implement policies on disposal of carcasses and subsequent disinfection and decontamination Control measures aim at breaking the cycle of infection. It is primarily around proper disposal of anthrax carcasses, disinfection, decontamination and disposal of contaminated materials, and vaccination of exposed susceptible animals and humans in at risk occupations. (Thomas G Hull. Disease Transmitted from Animals to Man, 5th ed, 1963: 82-125.) 9 Guidelines for an effective control programme Surveillance All unexplained livestock deaths or suspected cases must be investigated with laboratory support. Reporting Mandatory reporting of sudden deaths among livestock Mandatory reporting of all human cases Vaccination All high risk persons (veterinary workers, laboratory workers, workers handling potentially contaminated industrial raw materials) should be immunized. Annual boosters are necessary if exposure continues. Immunise all animals at risk and re- immunise annually. Disposal After confirmation as being a case of anthrax, a carcass should not be opened and should be disposed of by incineration or rendering. Deep burial after disinfection is a less favoured option. Blood from the dead animal should be collected aseptically for confirmation of diagnosis. Necropsy should not be done, as this has the risk of spread of the infection. Disinfection Disinfectants should be available in reasonable quantities at veterinary hospitals. Veterinary assistants, surgeons and livestock owners should be trained in their use. Decontaminate soil seeded by carcasses. Education Educate employees handling potentially contaminated articles about modes of anthrax transmission, care of skin abrasions and personal cleanliness. Control dust and properly ventilate all hazardous industries particularly which are handling raw animal materials. Do not use/sell hides of animals exposed to anthrax nor use their carcasses as food or feed supplements. Treat properly the effluents from hazardous industries handling animals etc. (Thomas G Hull. Disease Transmitted from Animals to Man, 5th ed, 1963: 82-125.) Treatment All symptomatic animals should be treated. Immunize after cessation of treatment. 10  Actions to be taken in the event of an outbreak of anthrax:  Every effort is to be made to investigate the outbreak, to confirm through laboratory diagnosis and to search for the source.  In the affected area, the following measures must be applied:  The carcasses of infected cattle are to be either burnt at the site of death and the ashes buried deeply, or wrapped in double thickness plastic bag to prevent spilling of body fluids and removed to a more suitable site where they are burnt and the ashes buried.  The site where the animal died is to be disinfected with 5% formaldehyde after disposal of the carcass.  All other animals in the affected herd are to be vaccinated.  Affected premises are to be quarantined for at least 20 days after the last case or 6 weeks after vaccination, whichever is later.  Any milk collected from a cow, buffalo or goat showing signs of anthrax within 8 hours of milking is to be destroyed, along with any other milk that may have been mixed with the suspected milk.  People entering infected premises are required to wear protective clothing and footwear, which are disinfected before leaving the premises.  All cattle on neighbouring premises should also be vaccinated.  A buffer zone, 20-30 Km wide, is to be established around the infected area within which all cattle and exposed sheep are vaccinated and quarantined.  Persons who have handled infected animals or their carcasses should be vaccinated against anthrax, if their exposure is frequent and if the human vaccine is available.  Such persons should avoid any contact with other persons or animals without first changing clothing, washing hands and taking appropriate disinfection measures.  Where there is a risk of aerosolization of spores, further precautions should be considered such as damping down the material, possibly with 5% formalin, wearing facemasks etc. (Thomas G Hull. Disease Transmitted from Animals to Man, 5th ed, 1963: 82-125.) 11  Anthrax vaccination: In humans Anthrax vaccine is indicated for individuals who come in contact in the workplace with animal hides, furs, bone meal, wool, animal hair (especially goat hair), and bristles; and for individuals engaged in diagnostic or investigational activities which may bring them into contact with anthrax spores. In animals Vaccination is the hub of anthrax control in endemic areas. Therefore, a contingency stock of a vaccine meeting acceptable standards should be available in such areas at all times. For maximum success, vaccination as a control measure must be applied together with other control measures and must be continued for a full specified period. 12  BRUCELLOSIS SYNONYMS:  In animals: Bang's disease, Contagious abortion, Epizootic abortion.  In humans: Melitococcosis (Brucella melitensis), Malta fever (Brucella melitensis), Mediterranean fever (Brucella melitensis), Undulant fever (Brucella abortus) INTRODUCTION: Brucellosis is one of the serious diseases affecting the livestock of our country. The incidence of the disease has been reported from various parts of India and it has been observed that the disease is more common in organised herds and farms in comparison to isolated animals. Cases of human brucellosis are frequently reported from various parts and although statistical data on human brucellosis are wanting, the disease could he considered a serious health risk considering the enormous cattle wealth that India is bestowed with. Brucella spp. are Gram negative, facultative intracellular pathogen, non motile, aerobic and coccobacilli  The "classic Brucella" are Brucella abortus in cattle, B. melitensis in goats and B. suis in swine. Other zoonotic Brucella are B. canis in dogs, B. ovis in sheep and B. neotomae in rats, particularly in desert rat. B. ovis and B. neotomae are nonzoonotic agents. Recently B. maris has been identified from marine animals.  Biovars of Brucella spp. o Brucella melitensis - Biovars 1 to 3 o Brucella abortus - Biovars 1 to 7 o Brucella suis - Biovars 1 to 5 As a global problem brucellosis commands all attention because of its public health and economic implications. In man it may take the form of a prolonged, often vague, illness requiring expensive therapy and may produce varying degrees of incapacitation even when the patient is ambulatory. In animals, serious losses occur due to abortions, still births premature birth of weaklings, infertility,loss of meat & milk. EPIDEMIOLOGY:  The disease is widely prevalent in all the countries of the world. Brucellosis is still a serious problem facing the Veterinary and Medical professions.  In India, the disease is widely prevalent in all the States. 13   Human brucellosis due to B.canis is uncommon but can be acquired from dogs; most cases resulted from contact with aborting bitches.  Each year about a half million cases of brucellosis occur around the world (WHO, 1975).  It has been eradicated from Finland, Norway, Sweden, Denmark, the Netherlands, Belgium, Switzerland, Germany, Australia, Hungary, the former Czechoslovakia, Rumania and Bulgaria.  Swine brucellosis is never had this problem in Muslim countries as a result of religious beliefs that have limited swine rising.  The natural reservoirs are cattle (Brucella abortus), goats (Brucella melitensis) and swine (Brucella suis). Socio economic impact:  Biological warfare: The United States biological warfare program focused on three agents of the Brucella group: Porcine Brucellosis (agent US), Bovine Brucellosis (agent AB) and Caprine Brucellosis (agent AM). Agents US and AB had a median infective dose of 500 organisms/person and AM was 300 organisms/person. The rate-of-action was believed to be 2 weeks, with duration of action of several months. AM was always believed to be a more virulent disease and a 3% fatality rate was expected.  Permanent sterility in male  Monetary loss  Reduced export on international trade  Loss of man-hours and man-days  Abortions in both animals and human beings. TRANSMISSION: The modes of transmission to man are ingestion, contact, inhalation and accidental inoculation. Infection by ingestion may occur via the gastrointestinal tract or by penetration through the mucous membrane of the throat. The usual vehicles of infection for man are: (a) untreated food products of raw milk origin from infected animals; (b) raw vegetables contaminated by infected animal urine or faeces; (c) viscera, bone marrow, and lymph nodes in muscle tissue from infected carcasses, which may retain viable brucellae for more than a month after slaughter, and for much longer in the case of frozen or chilled meats; (d) water supplies, such as cisterns and wells, contaminated by infected animal excreta. Contact with brucellae in vaginal discharges, foetuses, placentas, urine, manure, caracasses, and salvaged animals cause a large proportion of human cases. The skin and mucous membranes, including the conjunctivae, provide the portals of entry. The contact route of transmission is especially important among veterinarians, farmers, rendering-plant employees, packinghouse workers, animal handlers, factory 14 workers engaged in the primary processing of wool, and laboratory workers. The chances of infection by contact are particularly high during the season in which abortions occur, mainly because of the massive contamination of the premises. Infections by contact play an important role among those who, because of climatic conditions, bring their animals into the human habitation, where transmission of brucellae from animals to humans occurs, especially among children who use the animals as pets. Water shortages in summer may prevent adequate personal hygiene and thus tend to increase the possibility of the infection being transmitted to man, In winter, extremely cold temperatures may have similar results by bringing infected animals into closer contact with shepherds. Infection occurs when man inhales infected dried materials of animal origin, such as the dust from sheep wool, railway trucks and lorries that have transported infected animals, abattoirs, infected farm premises, and brucella laboratories.Infection by accidental inoculation is not frequent among veterinarians and laboratory workers. Those involved in the large-scale production of brucella vaccines and diagnostic antigens are also at special risk of infection and/or sensitization reactions. Spread of infection to urban areas may occur when urban dwellers purchase goats that have aborted and which are therefore, sold at reduced prices. In some countries, Br melitensis infections may occur especially in summer during periods of bovine milk shortages, since the milk of cows and buffaloes may then be mixed with the less popular, and frequently infected, milk of goats and sheep and consumed as raw milk or ice cream.\ The transmission of brucella infection to man and its prevalence in different areas of the world depend upon local food habits, methods of processing milk for cream, butter, and cheese, social customs, types of animal husbandary practices, species ofbrucella prevalent in the region, climatic conditions, and standards of personal and environmental hygiene. Clinical manifestations: HUMAN BRUCELLOSIS: The clinical manifestations of human brucellosis are variable. In an endemic area, the clinician must consider brucellosis in the differential diagnosis of any febrile disease. Acute and subacute brucellosis Acute and subacute brucellosis are accompanied by fever and bacteraemia. Some patients develop an acute illness of limited duration followed by apparent recovery; others have prolonged fever followed by frequent relapses. Either variety can be accompanied by various complications (articular, osseous. visceral, and neurological). 15 Chronic brucellosis Chronic brucellosis, which is usually non-bacteraemic, occurs with or without demonstrable localized foci of infection. The symptoms are generally related to the state of hypersensitivity of the patient. Illness may persist for a number of years. The disease's sequelae are highly variable and may include granulomatous hepatitis, arthritis, spondylitis, anemia, leukopenia, thrombocytopenia, meningitis, uveitis, optic neuritis and endocarditis. Disease in animals  The incubation period is ranged from 1 to 3 weeks, but some rare instances may take several months.  Abortions (at third trimester of pregnancy) are followed by immunity  Carrier state persists especially with secretions from the udder.  Infertility, testicular abnormalities and poor semen quality.  Mastitis.  Inapparent infection may be common, as indicated by seropositivity. Diagnosis: The diagnosis of human brucellosis is based on epidemiological information, clinical manifestations, and laboratory tests. Diagnostic methods include bacteriological, serological, and allergic tests. Bacteriological tests The tissues from which Brucella can most easily be isolated are blood and sternal marrow. If first attempts are unsuccessful they should be repeated. Other sources, such as lymph nodes, cerebrospinal fluid, urine, and any abscesses that maybe present, should be investigated. Brucellae have occasionally been isolated from sputum, placenta, mother’s milk, vaginal discharges, seminal fluid, etc. Serological tests These tests should be repeated when the first tests are suspicious or negative in cases of active disease. Serum agglutination test The agglutination test, when carried out with a suitable antigen and a satisfactory technique, nearly always gives significantly positive results in the presence of active infection. The agglutination test should be repeated when other serological tests carried out in cases giving low titres or negative reactions. In suspected cases, a high 16 or rising agglutination titre is presumptive evidence of brucella infection. High titres usually indicate infection, but low titres or negative reactions do not exclude it. Low titres are quite significant when the 2-mercaptoethanol tests show the presence of 7S immunoglobulin. It should be noted that a positive agglutination test may be produced by cholera, tularaemia. Cholera induced agglutinins for brucella can be differentiated by the agglutinin-absorption test and by the fact that treatment with 2- mercaptoethanol removes the cross-reacting substances. Attention is drawn to the existence, in some endemic areas, of large numbers of persons without symptoms but with low brucella agglutinin titres. In patients with ill- defined symptoms and low titres, the presence of specific 7S imrnunoglobulin is important. When antibiotics are freely used in febrile conditions, positive diagnosis by blood culture is less frequently possible, and increasing reliance is therefore placed on the results of agglutination and other serological tests on the clinical picture. Complement Fixation test This test also should be used in the diagnosis of both acute and chronic brucellosis. Coombs’ test This test is important for the detection of chronic cases. It is useful for epidemiological surveys of past brucellosis in endemic areas, and when active brucellosis is suspected but the serum agglutination test is negative. Other tests The passive (indirect) haemagglutination test can be used to diagnose brucellosis. It is specific and more sensitive than the agglutination test. The indirect immunofluorescence test is a specific, rapid, and sensitive method of detecting antibodies in human sera, and should be used in qualified laboratories. Limited experience has shown that the buffered Brucella antigen (card, Rose Bengal) test may be of value in diagnosis and deserves further evaluation. Intradermal test A positive intradermal test normally indicates a state of specific allergy for brucellosis. When possible, the test should be performed with an allergen that neither stimulates antibody formation nor produces a non-specific skin reaction. A positive intradermal test, in healthy persons with past exposure to infection, can result in a rise in agglutinins when certain skin-test allergens are used. The intradermal test is only a complementary aid in the diagnosis ofbrucellosis. It can be helpful : (a) in chronic brucellosis, when a positive test maybe the only objective indicator of infection; great caution should be exercised in making a diagnosis of chronic 17 brucellosis based solely on a positive skin-test and vague clinical symptoms, since a positive brucella skin reaction is known to occur at various stages of other chronic diseases; (b) when it is persistently negative; this excludes brucellosis; (c) in areas with a low incidence of endemic brucellosis, when a positive reaction may have important significance; (d) in epidemiological surveys; it is important to bear in mind the fact that vaccinated individuals may have positive skin-tests. TREATMENT: In the treatment ofbrucellosis, the acute and subacute forms must be considered separately from the chronic form of the disease. Therapy of acute and subacute brucellosis In view of the frequency of spontaneous recovery, the following points are of value injudging the effectiveness of any therapy: a) Clinical improvement within a week; b) The failure to recover brucella from the blood, or from other tissues or sites, when previous results were positive; c) A reduction in the frequency of complications; d) A reduction in the frequency of relapses; e) A drop in antibody titre. Therapy of chronic brucellosis In cases of persistent localized infection, treatment with antibiotics and even surgical intervention may be useful. However, desensitization should be considered. Physiotherapy may be useful adjunct to treatment. Prevention and control  Human brucellosis can be prevented by controlling and eradicating animal brucellosis.  Quarantine and testing.  Screening the herds and remove the reactors.  Test and slaughter method will be the most rational approach.  Hygienic disposal of aborted uterine discharges, foetus and foetal membranes.  Vaccination of all calves between 4 and 8 months of age with strain-19 vaccine (Dose is 5 ml in s/c route). 18   The main way of preventing brucellosis is by using fastidious hygiene in producing raw milk products or by pasteurization of all milk that is to be ingested by human beings, either in its pure form or as a derivate, such as cheese. Vaccination  Strain 19 vaccine: Live-attenuated vaccine strain.  RB51 vaccine: Newer live-attenuated vaccine strain that is used at present.  "Calfhood vaccination": Vaccination should be done during calfhood (4 to 8 months for S19; 4 to 12 months for RB51) so as to minimize the induction of antibodies that might be interpreted as evidence of actual infection.  Vaccination should not be conducted in pregnant animals because of the risk of vaccine-induced abortion.  S19 vaccine will induce brucellosis in humans who are inadvertently stuck. If this happens, person should receive doxycycline for 3 weeks and rifampicin antibiotics prophylactically. The potential for human disease due to RB51 remains unclear, but prophylaxis with doxycycline is prudent. 19  TUBERCULOSIS Synonyms  Phthisis ('Consumption' - Tissue wasting), Pearl disease (Grape-like lesions in the peritoneum and pleura), Scrofula (King's evil), Pott's disease (paralysis of the hind limbs due to TB nodules of the spine). Type of zoonosis  Direct anthropozoonosis, Zooanthroponosis Definition  It is a chronic disease of man and animals caused by pathogenic Mycobacterium spp. causing development of tubercle in vital organs. The pulmonary tuberculosis is the most common form, characterized by protective cough, fever, fatigue, weight loss, chest pain and night sweat in human beings. Brief history  Tuberculosis (TB) is an ancient disease known as early as 4000 B.C. Tuberculosis was well described by Hippocrates and Aristotle in the fifteen century BC. As ‘Phthisis’ (described in Greek literatures) which was translated in to English as ‘Consumption’.  Robert Koch in 1882, cultivated the agent and demonstrated tuberculin testing first in guinea pigs in 1890.  French scientists, Calmette and Guerin (veterinarian) in 1906, developed vaccine against TB, is known as "BCG vaccine". It was developed by attenuating a virulent strain of Mycobacterium bovis. They made 230 subcultures over a period of 13 years and evolved a strain known as "Bacille Calmette Guerin", which was avirulent for man while retaining its immunogenicity. In 1927, the first human was vaccinated and in 1948, the vaccine was accepted by TB workers.  Tuberculin test was first discovered by Von Pirquet in 1907.( Park.K., 2009. Park's Textbook of Social and Preventive Medicine, 20th edition, Banarasidas Bhanot Publishers, India.) 20 Etiology  Tuberculosis is caused by bacillus of the bacteria belong to the genus Mycobacterium. Mycobacterium organisms are facultative intracellular pathogens, Gram positive, acid fast (because Mycobacterium has mycolic acid in its cell wall) and aerobic. The most common zoonotic species of Mycobacteria are: o M.bovis - Cattle, dogs and swine o M.avium complex (MAC) - Birds, swine and sheep o M.tuberculosis - Man, nonhuman primates, cattle, dogs, swine and psittacines o M.marinum, M.fortuitum, M.platypolcitis - Fish  MAC is composed of 28 serotypes. MAC includes M.avium - intracelluare (MAI) and M.avium - intracellulare - scrofulaceum (MAIS). The disease produced by MAC complex is called as Mycobacteriosis in man. ( http://www.tnhealth.org/) Incidence  The disease is discovered more than 100 years ago. There are about 60 million cases of tuberculosis in the world. Around 5000 people die from tuberculosis daily. According to the estimates of WHO about one third of the total population of the world is infected with tubercle bacteria. This has made the disease an international health problem.  Morbidity and mortality rates continue to be higher among urbanites, minorities, the poor, the homeless, substance abusers and persons infected with HIV.  Global incidence is more in low income countries.  South-east Asia region countries carry 38% of the global burden of tuberculosis, with 3 million new cases and nearly 0.6 million deaths occurring every year.  Rising trend in HIV infection and emergence of multidrug resistant strains of TB (MDR-TB) pose additional threat.  The incidence of TB is influenced by may factors, are: o Poor or inadequate health care o Poor standard of living and socioeconomic conditions o Malnutrition o Higher population density o Occupational contraction o Poor personal hygiene 21  o Lack of education and awareness o Other diseases like, HIV/AIDS, Diabetes mellitus. o Close confinement of the human population.  The DOTS (Directly Observed Treatment, Short-course) strategy has been globally recognized as the best cost-effective approach to control TB and to reduce the disease burden and to reduce the spread of infection. DOTS, by which cure can be ensured.  Categorization of countries by DOTS strategy o "0" - Not reporting to WHO. o "1" - Not implementing the DOTS and case rate over 10 cases per 100000 populations. o "2" (Pilot phase) - Implementing the DOTS and case rate of 10% of the total population. o "3" (Expansion phase) - Implementing the DOTS in 10 to 90% of the total population. o "4" (Routine implementation) - Implementing the DOTS in over 90% of the total population. o "5" (Low incidence) - Not implementing the DOTS and case notification rate of 10 cases per 100000 populations.  As on 2002, 180 countries have implemented the DOTS, covering 69% of the world's population. In India:- o India globally ranks first in tuberculosis. o It is estimated that one-third of the current global population is infected asymptomatically with tuberculosis, of whom 5 to 10% will develop clinical disease during their lifetime. o India falls under MDR-TB zone. o TB is one of the biggest public health problems in India. As per the statistics, every year approximately 1.8 million people develop TB, of which 4.17 lakhs people die, that means, it was observed that every second an adult in India is infected with TB and every minute one Indian dies of this disease. o It is estimated that loss of an average about 83 work-days annually due to TB. o More burden in childhood and deaths from tuberculosis by meningitis and disseminated disease. o Since 1993, India has successfully implemented Revised National Tuberculosis Control Programme (RNTCP) using DOTS strategy. 22 Reservoir and source of infection  Human source: Sputum.  Bovine source: Milk and faeces.  Environmental source: Water, soil and dust contaminated with human and animal sources. Mycobacterium multiply in the water and soil.  Infected patients are infective as long as they remain untreated.  Effective treatment for TB reduces infectivity by 90% with in 48 hrs. Transmission  In human beings: o Ingestion and inhalation are the most common mode of transmission. o Mycobacterium bacilli are transmitted from infected animals or infected tissue primarily via the aerosol route. o Inhalation of droplets or droplet nuclei of sputum-positive patients, coughed up materials and fomites contaminated with droplets of sputum. o Contracted via ingestion or cutaneous inoculation of the bacilli. o Exposure to dusty bedding of infected animals, coughing of infected animals and aerosolization of the organism during sanitation procedures may also be sources of the disease in the laboratory environment. o Consumption of infected milk.( Park.K., 2009. Park's Textbook of Social and Preventive Medicine, 20th edition, Banarasidas Bhanot Publishers, India.)  In animals o Directly through contact with tuberculosis animals or their discharges or their sputum. o Contact with tuberculosis peoples. o Calves by ingestion of contaminated milk. o Congenital infection,Through artificial insemination with infected semen. (www.cdc.co.in) 23 Clinical signs  Disease in man o Incubation period ranges from 3 to 6 weeks, but uncertain. There after the development of disease depends upon the host-parasite relationship. It may take weeks or months or years. o The patient may be asymptomatic for years. General signs may include anorexia, weight loss, lassitude, fatigue, fever, chills and cachexia. o Primary tuberculosis is often asymptomatic. Basic sequence of events may be as (i). conversion of tuberculin sensitivity in 3 to 8 weeks, (ii). miliary and meningeal TB in 3 months, (iii). pleural effusion an pneumonia in 3 to 6 months and (iv). first appearance of post primary lesions in renal, bone and joints in one year. o In humans, the clinical signs depend on the organ system involved. o Chronic pulmonary TB: The most familiar signs related to pulmonary TB are cough, sputum production and hemoptysis. It is common in middle aged people who have preexisting pulmonary lesions. o Lymphadenitis: It is common in children from 18 months to 5 years of age. Lymph nodes are primarily those of the neck close to the jaw bone (cervical lymph nodes) affected. o Skin or cutaneous TB: Skin lesions are characterized by ulcers or by papular lesions progressing to dark suppurative lesions. o Miliary TB is most often seen in the very young and old people. o M.marinum of fish strain causes granulomas on the extremities as a group of papules that ulcerate and scab over, is described as "swimming pool granuloma or fish tank granuloma". Lesions may persist for months, and healing is usually spontaneous. Swimming pool granuloma may also be due to M.xenopi.(www.who.org)  Disease in animals o High fever and emaciation o Loss of body weight slowly o Difficult in breathing o Painful dry cough o Abdominal pain o Chronic bloat 24 Diagnosis  The diagnosis of TB is often difficult.  Clinical support with laboratory tests commonly used for presumptive diagnosis are: intradermal TB test with mammalian tuberculin, radiography, acid fast stained sputum smear (Ziehl-Neesen staining) and ELISA. Confirmation by culture, histopathology or animal inoculation.  Molecular diagnosis by polymerase chain reaction.  In humans o Tuberculin test: Mantoux intradermal test (most preferred), Heaf multiple puncture test and Tine multiple puncture test. o Heaf multiple puncture test for screening large population, quick and easy test, and reliable and cheap test. o Tine multiple puncture test is an unreliable test, and not recommended. o Mantoux intradermal test: Inject 1 ml of 1 TU of PPD-RT-23 with Tween 80 intadermally on the flexor surface of the forearm and then read the result after 72 hrs. Tuberculin reaction is erythema and induration at the site of injection. Reaction exceeding 10 mm is positive,