Haemorrhagic septicaemia

S S BASTIANELLO AND M M HENTON

GENERAL INTRODUCTION: FACULTATIVELY ANAEROBIC GRAM-NEGATIVE RODS Pasteurella and Mannheimia spp. infections

Introduction and aetiology

Haemorrhagic septicaemia is a peracute to acute, highly fatal bacterial disease, principally of cattle and water buffalo (Bubalus bubalis), in which Pasteurella multocida serotype B or E is the sole causal organism. It is endemic throughout south-east Asia, India and many regions of Africa.^{12, 16, 28} The Asian form is caused by serotype B and the African form (East African haemorrhagic fever) primarily by serotype E.

By means of an indirect haemagglutination test (Carter procedure) P. multocida strains are characterized according to their capsular antigens into five capsular groups (A, B, D, E and F) and, using a gel diffusion test, into 16 somatic types (1 to 16) (Heddlestone procedure).³⁹ Traditionally haemorrhagic septicaemia has been regarded as being caused only by serotype B:2 or E:2 but other strains are recognized as causing it, in particular types B:1 and B:3,4. Both of the latter types have been linked to outbreaks of haemorrhagic septicaemia in the USA whilst B:3,4 was involved in disease among reindeer (Rangifer tarandus) in Lapland and fallow deer (Dama dama) in Denmark and the UK,1, ³⁸ and serotype B:2 has been implicated in an outbreak in cattle in Zimbabwe.^{28, 33}

For further information on the characteristics of Pasteurella spp., consult the introduction to Pasteurella and Mannheimia spp. infections.

Epidemiology

Haemorrhagic septicaemia occurs commonly in several countries in western and eastern Africa, including Senegal, Mali, Ivory Coast, Nigeria, Cameroon, Egypt, Sudan, Congo, Democratic Republic of the Congo and Zambia, and has occasionally occurred in southern Africa in Namibia and Zimbabwe. ^{4, 6, 7, 20, 22, 28, 33, 34, 35, 36, 46} In Asia the disease is especially prevalent in south-east Asia including Indonesia, Philippines, Malaysia and Thailand as well as in India and Sri Lanka.^{12, 16, 17, 30} It has on rare occasions also been recorded in the USA and Canada where in both countries bison (Bison bison) were principally affected.^{27, 31} The disease has also been reported in cattle in Argentina, southern Europe and Russia^{6, 37} and, recently, in cattle, pronghorn (Antilocapra americana) and elk (Alces alces) in the USA and reindeer in Lapland.³⁸

Until recently type E was believed to have been the cause of haemorrhagic septicaemia in all countries in Africa except Egypt and Sudan where both serotype B and E have been implicated8, ^{22, 25} possibly because of the proximity of these two countries to Asia, where only serotype B is involved. Recent outbreaks of the disease in Namibia and Cameroon, however, have been caused by serotype B:2, whilst an outbreak in Zimbabwe in 1990 was caused by B:2.^{20, 32, 47}

In Africa haemorrhagic septicaemia is endemic in the tropical and subtropical regions of several countries in west Africa and the horn of Africa. Outbreaks in these parts tend to be sporadic and may spread to the drier and semi-arid regions of the Sahel when cattle are subjected to stress such as that induced by abnormally wet weather patterns or their migration to other parts.^{22, 36} In southern Africa the disease is very sporadic and mainly restricted to the wetter subtropical regions of Zambia, Zimbabwe and the northern parts of Namibia. In the latter country it occurs particularly in the Caprivi, Ovamboland and, to a lesser extent, Otjiwarongo, Okahandja, Gobabis and Grootfontein districts, although it has also been reported from drier areas such as the Keetmanshoop and Windhoek districts.^{7, 9, 20, 28, 47} The Zimbabwe outbreak, which was severe, occurred in the unusually wet and cold summer of 1990 on a farm irrigated with sewage effluent.²⁸ There is only one record of the disease occurring in South Africa where it involved calves in the relatively dry but hot North West Province.⁸

The Asian form of haemorrhagic septicaemia occurs in countries with a high seasonal rainfall where it is frequently endemic in marshy zones or along river deltas.³⁶

In the endemic regions of both Asia and Africa, haemorrhagic septicaemia occurs throughout the year but its prevalence increases at the onset of, or during, the rainy season when it tends to spread, presumably due to a longer survival time of the organism in damp conditions.^{4, 5, 17, 19, 22} It has been reported to occur following a sudden increase in rainfall and humidity with a concomitant severe drop in temperature. ⁴ The outbreaks which occurred in Namibia in 1994 and 1995 were not associated with increased environmental moisture but took place in winter when the affected cattle were subjected to severe cold and stress associated with confinement.⁴⁷

Under natural conditions, haemorrhagic septicaemia occurs mainly in cattle and water buffalo but it has also been reported in pigs, sheep, horses, donkeys, camels, fallow deer, bison and Indian elephant (Elephas sengalensis).^{3, 7, 12} Water buffalo are more susceptible than cattle,^{12, 24} the mortality rate in Sri Lanka being three times greater in water buffalo than in cattle.¹⁶ The disease has been reported in goats in India and Malaysia,16 but in Sri Lanka they were found to be highly resistant to experimental infection; after being inoculated with large numbers of the causative organism they developed neither the disease nor any detectable immunity. ^{16, 17} In endemic regions the disease usually affects the calves of cattle or water buffalo between six months and two years of age, with an annual morbidity rate in this age group being 30 to 32 per cent.¹⁷ It is seldom encountered in animals older than four years, but, when it does occur in animals of this age group, the morbidity rate is 3 to 5 per cent for cattle and 8 to 9 per cent for water buffalo.^{15, 17, 18} In nonendemic regions, animals of all age groups are affected and both morbidity and mortality rates are high.^{16, 17, 19, 27} In Asia the prevalence is generally higher in large, free-range herds than in smaller herds in which the management level is generally of a higher standard.¹⁶

Depending on various factors, such as the level of infection, intercurrent disease and immune status, some animals will succumb to the effects of the disease whilst others develop a so-called arrested infection. Animals in the latter category are latent carriers and possess high antibody levels. ^{16, 17} Carrier animals serve as a source of infection and play an important role in the epidemiology of the disease.^{8, 12, 16, 34} In a herd recently exposed to infection up to 23 per cent of the animals may be carriers, P. multocida being harboured in the nasopharynx, retropharyngeal lymph nodes and/or tonsils.^{17, 40} Animals can be active or latent carriers. In the former the bacteria are located in the nasopharynx and tonsils and excreted but in the latter they are present only in the tonsils and are not shed. Studies of the tonsils using an immunohistological peroxidase technique have shown that P. multocida is located within the tonsillar crypts; this explains why attempts to clear carrier animals of infection by administration of antibiotic therapy fail.^{16, 17, 25} The active carrier state is short-lived, lasting four to six weeks,¹² whilst the latent state can be more prolonged. Latent carriers can become active carriers on an intermittent basis following the intervention of one or more stress factors such as intercurrent protozoal or other bacterial infections, transport over long distances, starvation, overcrowding and administration of intramuscular injections.^{4, 8, 16, 27, 40} An outbreak of haemorrhagic septicaemia in Namibia in 1994/95 was associated with a period during which the cattle concerned were subjected to multiple stress factors, namely cold weather, poor physical condition attributable to feed shortages associated with an extended drought, prolonged ‘yarding’ for drenching, branding, castration and vaccination purposes, and prolonged contact with oryx antelope (Oryx gazella) which are speculated to be latent carriers of P. multocida B:2.⁴⁷

An outbreak occurs when a latent carrier becomes an active one and commences to shed bacteria which infect in-contact susceptible animals, leading to the establishment of overt disease and an escalation in the rate of dissemination of the infection.¹⁶ The rate at which the disease spreads depends on the proportion of naive animals in the herd.¹⁶

Pasteurella multocida is excreted in the respiratory aerosols, saliva, urine, faeces and milk of infected animals.^{3, 12, 29} Transmission of the infection is either by direct contact between affected and susceptible animals or by indirect means such as infected fomites or aerosols. Experimentally, animals can be infected via the subcutaneous, intranasal, per os or intramuscular routes.^{12, 16, 17}

The magnitude of an outbreak of haemorrhagic septicaemia is dependent upon the level of immunity in the animal population.¹² An explosive outbreak with high morbidity and mortality rates can be expected when an animal that is shedding P. multocida is introduced into a herd in which the animals possess no or minimal immunity. On the other hand, in endemic areas where large proportions of adult animals are immune, the disease tends to be restricted to young animals that possess declining levels of passive colostrum-derived immunity or to recently introduced animals from non-endemic areas. Mortality rates can vary from 1 to 10 per cent in endemic regions and 20 to 98 per cent in non-endemic regions.^{12, 19}

Pathogenesis

In natural cases P. multocida probably enters an animal via the nasal and/or oral routes and initially multiplies in the tonsils. Fever, salivation and dullness occur a few hours after infection. Bacteraemia has been detected within 12 hours of experimental infection and at this stage the organisms are usually present in the saliva.¹⁶

The effects of the infection are dependent upon the virulence and number of infecting organisms and the susceptibility of the host.¹² The virulence of the organism is associated with the presence of capsules; virulent organisms producing smooth, iridescent colonies on culture.^{12, 29} Overwhelming of the defence mechanisms of the host by the organism results in septicaemia and overt disease.¹² There is mounting evidence that endotoxins in the bacterial cell wall are responsible for the lethal effects of the infection.^{12, 37}

As the disease progresses, affected animals may show respiratory distress and a reluctance to move and may become recumbent. At this stage the venous blood pressure falls and P. multocida appear in the faeces, urine and milk.¹²

Clinical signs

The incubation period of the natural disease is two to three days but may be as short as 30 hours in experimentally induced infections.¹² The disease may run a peracute, acute or subacute course. The peracute disease is characterized by sudden death, generally with no premonitory signs being noticed, but some animals may manifest dyspnoea, grunting and prostration for a few hours to 24 hours prior to death.^{8, 27, 29} Acutely and subacutely affected animals may be pyrexic and exhibit anorexia, depression, profuse salivation and nasal discharge, with rapid respiratory and pulse rates prior to death.^{12, 22}

Subacute disease is characterized by the development of a firm, subcutaneous swelling of the submandibular region which may extend caudally to involve the subcutaneous tissues of the neck, brisket and fore legs.^{3, 7, 8, 12, 26} Some animals may show circling movements and incoordination.⁷ Clinical signs are generally present for periods ranging from eight to ten days.^{7, 8, 27} The disease is generally fatal if animals are not treated.^{26, 27}

Pathology

Peracute and acute haemorrhagic septicaemia is usually characterized by severe generalized congestion and widespread petechiae and ecchymoses of the serosal surfaces and subcutis, abomasal suggillations, mild to moderate ascites, hydrothorax and hydropericardium, focal anasarca and hyperaemia at the thoracic inlet, and lymph nodal oedema, congestion and petechiation.^{8, 12, 27, 37} Some animals may show severe oedema of the lungs or a focal fibrinopurulent pneumonia, oedema and haemorrhages of the tracheal mucosa, adventitia, and peritracheal connective tissue, and localized serofibrinous myositis of one or more muscles.^{8, 37} Microscopically, the acute disease is characterized by multisystemic hyperaemia and leukocytosis and a localized serofibrinous to necrotizing myositis evidenced by myofibril necrosis and fragmentation with interstitial oedema, haemorrhage and serofibrinous exudation.^{8, 24, 27} Animals infected experimentally by subcutaneous inoculation develop extensive oedema at the injection site²⁴ containing large numbers of bacteria which are also present in draining lymph nodes. In naturally infected cattle, bacterial emboli are frequently noted in pulmonary lymphatics and, occasionally, blood vessels of other organs and tissues, whilst, in water buffalo they are usually encountered in the blood vessels of the adrenal glands and spleen.²⁴ In animals that have died from the disease bacteria can, with the aid of the immunoperoxidase technique, be seen in both blood vessels and lymphatics in the lungs as well as in alveolar macrophages, while in other tissues they are only evident in blood vessels emphasizing the terminal septicaemic nature of the acute disease.²⁵

Subacute disease is characterized by severe localized cellulitis, fasciitis and myositis with micro-abscessation and fibroplasia of the subcutis of the submandibular region, neck, brisket and/or fore legs and lymphadenitis of the draining lymph nodes.^{4, 7, 8} Other lesions may include generalized congestion, widespread serosal haemorrhages, pulmonary oedema, accumulation of serofibrinous or blood-stained fluid in the thoracic cavity, focal fibrinous pleuropericarditis, serofibrinous polyarthritis and tendosynovitis, myocarditis, myocardial and/or renal infarction, pseudomembranous erosive or ulcerative rhinitis and serofibrinous meningitis.^{7, 8} In cattle fibrin thrombi and bacteria are frequently encountered in lymphatics and venules of the affected areas and draining lymph nodes.^{8, 24}

Diagnosis

A presumptive diagnosis of haemorrhagic septicaemia can be made on the epidemiology, clinical signs and pathology, and by the demonstration of Pasteurella organisms in blood or tissue smears or organ sections stained by the Giemsa or Gram methods.¹² A definitive diagnosis rests on the isolation of the causative organism. Blood from live animals is seldom useful for this purpose as septicaemia often only develops terminally immediately before death.¹⁶ Specimens of blood from dead animals, or, in the case of those that have been dead for some time, of bone marrow should be submitted to a laboratory for isolation of P. multocida.¹⁶

Various methods have been employed to assign the pasteurellas to a specific biotype or serotype. For P. multocida, serotyping is achieved by characterization of the lipopolysaccharide capsular antigens and somatic protein antigens. In South Africa, typing is performed by identification of the carbohydrate capsular antigens by the Carter method of an indirect haemagglutination test, and the somatic antigens by the Heddlestone method of a gel diffusion precipitation test.³⁹ Further details of the methods employed can be found in the introduction to Pasteurella and Mannheimia spp. infections. An enzyme-linked immunosorbent assay (ELISA) for the serological identification of the haemorrhagic septicaemia strains is available.^{13, 39} This is a rapid and highly sensitive and specific serological test but a small percentage of haemorrhagic septicaemia-causing strains are negative with this ELISA. Immuno-electron microscopy has revealed that the ELISA-negative strains are a mixture of phenotypes with only 2 to 10 per cent of the population of these strains expressing haemorrhagic septicaemia- associated epitopes.⁴⁵ Work is underway to modify the haemorrhagic septicaemia ELISA to overcome this problem.¹⁴ Polymerase chain reaction (PCR), DNA fingerprinting and ribotyping are increasingly being used in an attempt to provide improved methods of diagnosis.³⁹

Ribotyping, a technique which utilizes labelled ribosomal RNA probes to highlight specific profiles generated by restriction endonucleases^{2, 39} has identified several ribotypes for P. multocida, each associated with a specific geographic region of origin and to a lesser extent the host species of the P. multocida. Two main ribotypes are recognized, ribotype 1 encompassing the North American B strains and the African E strains, and ribotype 2 encompassing the Asian strains.⁴² Pulsed field gel electrophoresis has revealed DNA profiles which are unique for types B, D and E strains of P. multocida and the PCR has identified a clone specific for the Carter B:2 haemorrhagic septicaemia- causing isolates.^{>43, 44, 45, 46}

Differential diagnosis

Clinically, acute haemorrhagic septicaemia should be differentiated from causes of acute mortality which include bacterial septicaemias and toxaemias such as those caused by Bacillus anthracis, enterobacteria, clostridia and other Pasteurella spp. as well as acute chemical and plant intoxications, in particular arsenic poisoning. The subacute form may be confused with conditions in which submandibular, peripharyngeal or brisket oedema occur, such as calf diphtheria, verminosis, snake bite, dosing injuries and congestive heart failure.

Control

The acute nature of most cases of the disease limits the efficacy of antimicrobial therapy and once signs are apparent, therapy is of little value.^{12, 16, 22} Antibiotic and sulphonamide resistance in haemorrhagic septicaemia-causing pasteurellas has not been reported and an outbreak can be effectively controlled by the administration of a sulphonamide (e.g. sulphadimidine or sulphamethazine) or an antibiotic (e.g. oxytetracycline, penicillin, ampicillin or chloramphenicol) to all in-contact animals that reveal a febrile reaction.^{12, 16}

Immunity may be actively acquired through natural exposure or vaccination, or in the new-born animals, passively, following ingestion of colostrum from immune dams.¹⁵ Solid, long-lasting immunity is conferred on animals that recover from the natural disease.¹² In many Asian countries where the disease is endemic, vaccination is the principal means of control and has been effective in decreasing the prevalence of the disease^{.3, 16, 19, 23} The vaccines used are inactivated vaccines and are generally

produced locally from local strains.¹⁶ Alum precipitated and aluminium hydroxide gel vaccines are the most widely used vaccines in Asia despite the fact that the immunity induced by them is only of four to six months’ duration whereas oil adjuvant vaccines confer an immunity for periods of 9 to 12 months but are thick and viscous and hence difficult to administer. ¹⁶ In most countries in which alum precipitated vaccines are used, a policy of annual vaccination immediately prior to the rainy season is adopted, but in some countries, such as Thailand, twice yearly vaccination is practised.¹⁶ Current recommendations for the use of alum precipitated vaccines include the vaccination of calves at four to six months of age followed by the administration of a booster three months later. Thereafter animals receive an annual inoculation.¹² Vaccination with an oil adjuvant vaccine is practised on an annual basis in Sri Lanka, Malaysia, Indonesia, Iraq and Egypt.^{12, 16, 34} Outbreaks of haemorrhagic septicaemia have been brought under control in certain Indonesian islands where annual vaccination of at least 80 per cent of the susceptible population is achieved.⁴¹ As capsular antigens from a number of species of bacteria including the pasteurellae have been shown to be virulence determinants, vaccines produced in future for the control of haemorrhagic septicaemia may be comprised of genetically engineered acapsular strains of the organism. Preliminary investigations with P. multocida B:2 have shown that immunization with high doses of acapsular bacteria provides significant protection against wild type challenge.¹⁰ The active mouse protection test on its own is not a reliable indicator of the level of immunity induced in cattle as some vaccines which produce a good immunity in mice¹¹ do not result in the development of an adequate immunity in cattle.¹⁶

Vaccination of cattle is undertaken in several northern African countries.^{16, 32} In Cameroon an inactivated P. multocida 6:E (E:2) vaccine is used, but in view of recent outbreaks of haemorrhagic septicaemia in the country being caused by B serotypes, vaccination with bivalent vaccines is advisable.³² In African countries south of the equator specific haemorrhagic septicaemia vaccines are not generally available. An outbreak of the disease in Zambia was eventually controlled by the use of a formalinized vaccine obtained from Sudan.²² In South Africa, a combined bacterin/alum precipitated vaccine for cattle which contains P. multocida serotypes A, D and E and Mannheimia (Pasteurella) haemolytica offers some protection against haemorrhagic septicaemia caused by serotype B:2.^{21, 47}

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