Posts Tagged ‘ vaccine ’

Bacterial Pathogens of the Respiratory Tract

Mannheimia haemolytica

M. haemolytica is responsible for causing contagious bovine pleuropneumonia, a bacterial disease which causes pneumonia and inflammation of the lung membranes. It is a Gram-negative coccobacillus (elongated, rod spheres) which shows mild haemolysis when plated on blood agar plates. This species comprises of 12 capsular serotypes (of which some are more responsible for disease than others). Serotype A2 is associated with sheep pneumonia.

Typically, diseases associated with M. haemolytica present themselves as fever, along with nasal discharge, coughing, inappetance and weight loss. Death associated with this bacteria is typically due to acute fibrous pleuropneumonia. Pathological observations would reveal an obstruction of the bronchioles with a fibrous exudate and an accumulation of neutrophils and fibrin in the alveoli. There will also be thrombosis and distension of the lymphatic vessels.

Mannheimia haemolytica as an Opportunistic Pathogen

M. haemolytica resides in the nasopharynx and tonsils of healthy cattle. A balance between the host and bacteria, under good environmental conditions maintains homeostasis between the host and bacteria (i.e. no disease). Should environmental conditions alter however, the balance may be tipped in favour of the bacteria, removing the status of homeostasis and resulting in disease. Key inciting events for disease include; weaning, adverse weather conditions, dehorning, feed changes and transportation i.e. causing stress. Stress provokes the bacteria in the nasal/tonsillar mucosa allowing them to be inhaled in to the lungs.

In healthy cattle clearance of the bacteria from the lungs is efficient enough to prevent disease. This suggests that stress induced alterations in immune functions can lead to host susceptibility and thus development of disease.

Close contact with other animals increases exposure to microorganisms which upsets the natural balance of commensal bacteria within the host. Changes in the natural microflora can cause M. haemolytica to revert to a pathogenic state. Slight changes in the environment are responsible for creating a favourable situation for M. haemolytica to colonies the lung and thus cause disease.

Mannheimia haemolytica Virulence Factors

Virulence factors of M. haemolytica include:

  • Adhesins – Required for initial colonization and adherence to host cells
  • Capsular polysaccharide – Helps prevent opsonisation and thus has anti-phagocytic properties
  • Sialoglycoprotease – An enzyme which cleaves IgG and thus reduces opsonisation of the bacteria
  • Neuraminidase – An enzyme which reduces the viscosity of respiratory mucus, reducing the chance of the bacteria being excreted via mucus
  • Iron-binding proteins – Removes iron from the host, for the benefit of the bacterium
  • Leukotoxin – A primary toxin involved in the pathogenesis of M. haemolytica

Mannheimia haemolytica Leukotoxin

Leukotoxin (LKT) is an actively secreted exotoxin which targets leukocytes, primarily neutrophils. The toxin is encoded for by four genes (lkt A-D) which can be found in the RTX toxin operon.

  • lktA encodes for the active toxin
  • lktB and lktD encode for the secretion of the toxin
  • lktC encodes for proteins responsible in the transportation and activation of the toxin

LKT help bacteria survive by allowing them to evade phagocytes. It is a type 2 exotoxin (membrane-damaging) which binds to leukocytes via the cell surface receptor CD18 (an integrin). It forms pores in the cell membrane of leukocytes which leads to an influx of K+/Ca2+ ions. This promotes swelling of the leukocyte and ultimately lysis of the cell.

Neutrophils affected by the toxin also become overly active, they overproduce certain mediators, reactive oxygen species and proteases, all of which promote further cellular and tissue damage within the host. Lesions develop which are filled with fibrous exudate and thrombosis of lymphatic vessels occurs. The alveolar epithelium also becomes damaged which is believed to be associated with neutrophil infiltration.

Controlling Mannheimia haemolytica Infections

Methods which could be implemented to reduce cases of M. haemolytica infections include:

  • Livestock management – Reduce stress of cattle by effectively managing when and how calves are weaned, sold and transported
  • Antibiotic use – Injectable antibiotic regimes are extensively used, especially in the intensive farming seen in large feedlots. However evidence shows that M. haemolytica are developing resistance to many of the common antibiotics used, such as; penicillin, ampicillin, tetracycline and sulphonamide
  • Vaccines – Both killed and live attenuated vaccines are used as well as cell-free supernatants such as the leukotoxin or capsular polysaccharide
  • Genetically Modified plants – A suggestion has been made for the use of GM crops to feed cattle in the future. The GM plants would produce M. haemolytica antigens which would essentially act as edible vaccines

These control methods could help to restore balance to the situation (previously unbalanced by negative environmental factors). Thus homeostasis between host and bacteria would be restored and disease prevented.

Bordetella bronchiseptica

Bordetella bronchiseptica is an evolutionary progenitor of B. pertussis and is one of the organisms responsible for causing kennel cough in dogs. Kennel cough is the term used to describe a disease which causes coughing in a dog due to inflammation of the trachea and lower airways. Although kennel cough is primarily caused by infection of the airways with B. bronchiseptica it can also be caused by viruses such as canine parainfluenza. Clinical signs of kennel cough include; an intense cough, mucus, nasal discharge and breathing difficulties.

Kennel cough gets its name from the frequency of infections which arise in dogs temporarily kenneled for example whilst the owner is on holiday. A number of dogs can carry the infection and the close contact in the kennels promotes transfer of the infection. Infection is caused either airborne transmission or via direct contact and has an incubation period of 3-10 days. Infected dogs can carry and shed the infection for up to four months after recovering from the disease. Because it require a only a few B. bronchiseptica bacteria to establish and infection in the airways (i.e. low infectious dose), B. bronchiseptica are considered highly infectious.

Bordetella Bronchiseptica Virulence Factors

Adhesins

  • Fimbriae – Associated with the initial adherence of the bacteria to epithelia. This form of adhesin allows the bacteria to latch on to host cells and begin to proliferate and form infectious colonies.
  • Filamentous Haemagglutinin Adhesin (FHA) – A large, filamentous protein which serves as a dominant attachment factor for adherence to host colliery epithelia cells of the respiratory tract. It is associated with biofilm formation and possesses at least four binding domains which can bind to different cell receptors on the epithelial cell surface.
  • Pertactin – An ‘autotransporter protein’ capable of getting to the cell surface without the need of accessory proteins. Pertactin also acts as an adhesin.

Adenylate Cyclase (cyaA)

  • Structurally similar to M. haemolytica leukotoxin
  • Different function, cyaA enters host cells and catalyses large amounts of cyclic AMP
  • This disrupts cell signaling pathways, impairing cellular function and inducing apoptosis whilst allowing the bacterium to avoid phagocytosis
  • It also inhibits the expression of interleukin 12 (IL12) an inflammatory cytokine

Dermonecrotic Toxin

  • A type 3 exotoxin which acts intracellularly
  • Precise mechanism is unknown however the overall effects on the cell include; actin reorganisation, increased DNA synthesis and binulcleation
  • Its overall contribution to the pathogenesis of B. bronchiseptica is currently unknown

Not all these virulence factors are active at the same time however, only some are active depending on the temperature. At 37˚C all are active except the flagellum, at at 27˚C none are active except the flagellum. What this shows is that there is a global regulation of gene expression depending on the requirements of the bacteria’s survival.

For example, when B. bronchiseptica is required to colonise the respiratory tract (indicated by a 37˚C temperature i.e. body temperature) it is not going to need to use its flagella for movement so the genes encoding for flagella motion are not expressed. In contrast when B. bronchiseptica is outside of its target destination e.g. the nasal passageway (indicated by a lower temperature) the flagella genes are expressed again as movement will be required to reach the target destination (the airways). Genes encoding for virulence factors will not be expressed as there is no need when the bacterium is not at its target site of pathogenesis.

Treatment of Kennel Cough

Kennel cough is susceptible to many antibiotics (it is however resistant to erythromycin). There are bivalent canine vaccines available which vaccinate against B. bronchiseptica and canine parainfluenza virus. Many of B. bronchiseptica outer proteins are highly immunogenic which makes good vaccines (e.g. fimbriae, FHA, pertactin)

In Summary

M. haemolytica and B. bronchiseptica are two important veterinary pathogens as they both inhabit the same ‘niche’. This is interesting because they also share similar virulence factors. Both diseases result directly from the disruption of the respiratory epithelium and from immunopathology and both diseases are often polymicrobial, involving mutliple agents.

Diagnosis relies on isolation and cultivation of bacteria on agar plates after which the correct antibiotic treatment can be given. Vaccinations are available for both diseases.

These diseases show how the management of animals can influence disease epidemiology.

Leptospirosis

Introduction

Leptospirosis is a zoonotic disease caused by the bacterial genus Leptospira. Leptospires are spirochetes, a group of Gram-negative bacteria with long, thin, spiral structures and an internal flagella used for movement. The size of a typical leptospire is around 0.1μm wide and 6-20μm long. This narrow, helical structure enables them to burrow in to tissues, within tissues they may adopt a more spherical or granular appearance.

Etiology

The primary pathogenic strain of Leptospira is Leptospira interrogans, however there are also non-pathogenic strains such as Leptospira biflexa which is an environmental saprophyte (i.e. consumes dead organic matter).There are currently around >16 species of identified Leptospira.

Serovars & Serogroups

Serovars are groups of organisms, categorised depending on the antigens they present on their surface. Therefore if a number of organisms within a Leptospira species share the same antigens on their surface, they will be grouped together into one serovar. Differences in surface antigens occur within the same species for example, within the pathogenic L. interrogans species, around >250 serovars have been identified worldwide.

Serogroups are clusters of serovars, as there may be hundred of serovars within a species, it is useful to group those together which share similar properties. Important serovars of L. interrogans include:

  • Canicola (Primary reservoir host – Dog)
  • Icterohaemorrhagiae – (Rat, mouse)
  • Bratislava – (Rat, pig, horse)
  • Pomona – (Cattle, pig, skunk)
  • Grippotyphosa – (Rodents)
  • Hardjo – (Cattle)

Each serovar is not limited to its primary reservoir host however. They be transmitted to incidental hosts fairly easily. Incidental hosts include humans, dogs and cats as well as other domesticated animals.

Different serovars are also responsible for different clinical conditions which can range from abortion to haemorrhagic disease. An individual serovar may also cause different clinical conditions in different species, for example L.interrogans serovar hardjo causes abortion and still births in cattle, but in humans it can cause an influenza-like illness or liver/kidney diseases.

Epidemiology

Transmission Cycle

The transmission cycle of a typically Leptospira species is as follows:

  • Rodents shed Leptospira in their urine
  • Direct transmission of Leptospira to humans may occur at this stage
  • The urine contaminates the environment (e.g. soil, water) with Leptospira
  • Indirect transmission to humans may occur at this stage
  • Leptospira may be transmitted to other domestic animals via the environment
  • These animals may become infected and can shed Leptospira in their urine which can lead to direct transmission to humans or contaminate the environment as before

Rodents can acquire Leptospira from the urine contaminated environment, thus creating a cycle of transmission

Transmission via direct contact usually occurs by urine which contains the Leptospira organisms. However direct transmission may also occur via veneral or placental transfer as well as bite wounds or the ingestion of infected tissue material. Crowding of animals (such as in kennels or intensive farming) will enhance transmission of Leptospira. Animals which recover from the disease, may still be infected, thus making them carriers of Leptospira which can still be excreted chronically in their urine. This continues the spread of infection

Transmission via indirect contact can also occur. Methods of indirect transmission generally requires exposure to contaminated sources such as; soil, food, bedding or water sources. The bacteria enters susceptible hosts from the contaminated source via damaged skin or exposed mucous membranes such as in the nose, mouth, eyes etc. Leptospira remains viable in the environment (still able to cause infection) for months, this further enhances transmission.

Environmental Factors

The optimal habitat for Leptospira depends on their environment, if aquatic, optimal conditions are stagnant or slow moving waters. If terrestrial, a neutral or slightly alkaline soil pH is preferred. However, organisms may survive transiently in undiluted acidic urine. A typical temperature range of 0-25C is preferred, this often leads to seasonal fluctuations in the incidence of Leptospirosis.

Pathogenesis

Leptospira enters the host by penetrating mucous membranes via vunerable areas such as damaged skin, eyes, nose or the mouth. Their helical shape and flagella aids in tissue penetration. Upon entering the blood system, they begin to multiply rapidly. The presence of bacteria in the blood is called bacteraemia. They are then distributed around the body via the blood stream.

Once distributed around the body, they then further replicate in target organs and tissues (including the kidney, liver, spleen, central nervous system, eyes and genital tract). The incubation period is around 7 days, this factor depends on the species and the strength of the host immune system however.

The initial immune response will usually remove all Leptospira organisms from the blood and tissues but some will persist in the kidney tubules where they can continue to replicate. The Leptospira organisms in the kidney tubules manage to evade the host immune response by avoiding phagocytosis.

The damage done to the host’s organs and tissues is variable and depends on the virulence of the Leptospira serovar and how susceptible the host immune system is. The most serious of diseases occur in the incidental hosts, i.e. not the primary reservoir host.

Overview of Pathogenesis

Diagnosis

At present there are three different methods of leptospirosis diagnosis:

  • Detect leptospire antigens – Leptospire antigens will induce agglutination of antibodies. This can be tested using a microscopic agglutination test (MAT).
  • Isolation of Leptospires – Leptospires are isolated from the urine or infected tissues. However this can be very labour and time intensive as Leptospira species are slow to culture using growth medium, meaning it can take weeks before a positive/negative result is returned. Despite this, this method of diagnosis is probably the most reliable.
  • Polymerase Chain Reaction (PCR) – Molecular methods of diagnosis (such as PCR) are gaining popularity for diagnosing Leptospirosis, however PCR is unable to distinguish between serovars.

Prevention

For dogs there are currently two forms of vaccine available:

  • In the UK, a bivalent vaccine is used which protects against two serovars – canicola and icterohaemorrhagiae.
  • In the USA however, a quadrivalent vaccine is used, this protects against four serovars – grippotyphosa and pomona as well the canicola and icterohaemorrhagiae which the bivalent vaccine covers.

Some preventative measures are also being taken in cattle to protect against Leptospira borgpetersenii serovar hardjo.

The widespread use of these bivalent vaccines may be responsible for the observed decline in classic canine Leptospirosis infections, however this vaccine does not provide cover for other serovars.

In the USA canine leptospirosis has been classified as a re-emerging disease due to the increasing amounts of newly diagnosed cases. This may be due to the prevalence of grippotyphosa, pomona and bratislava in wild reservoir species which are spreading Leptospira through the domesticated animal population. This is good reasoning behind the introduction of the quadrivalent vaccine as it protects against these serovars (not bratislava, however cases of bratislava are low).

In the UK, rural cases of canine leptospirosis are greater than urban cases, possibly hinting at a greater transmission via wildlife. This is a breakdown of serovar cases diagnosed in the UK:

  • 60% – L. icterohaemorrhagiae
  • 20% – L. canicola
  • 6% – L. icterohaemorrhagiae copenhageni
  • 1.3% – L. bratislava