Streptococcus pneumoniae- Morphology and Cultural Characteristics, Pathogenesis, Laboratory diagnosis, Epidemiology, Prevention and control of diseases

 S. pneumoniae

• Gram positive, lanceolate shaped diplococcus - 

• Pneumococci 

•  Formerly called, Diplococcus pneumoniae 

•  Normal inhabitants of the human upper respiratory tract 

• Common cause of Pneumonia and Otitis media in children 

•  Also cause Sinusitis, Bronchitis, Bacteremia, Meningitis 

• First noticed by Louis Pasteur and Sternberg- inoculated with human saliva in rabbits and produced fatal septicemia- isolated pneumococci from the blood of infected rabbits- 

• Fraenkel and Weichselbaum established the relation between pneumonia and pneumococci

MORPHOLOGY 

-Small, slightly elongated cocci - One end broad or rounded and the other pointed 

- Flame shamed or lanceolate Occur in pairs with broad ends in apposition 

-Capsulated- a pair in a capsule- capsule best seen in material taken from exudates and maybe lost on repeated subcultures 

-Seen as more rounded in culture- Typical morphology may not occur 

- Non motile, Non spore forming 

-Gram positive-readily stained with aniline dyes

- Capsule demonstrated in India ink preparation 

CULTURAL CHARACTERISTICS 

 Complex growth requirements-Enriched media 

- Aerobes and facultative anaerobes 

- On Blood agar, at 18 hours, colonies are small (0.5 – 1 mm), dome shaped and glistening with α haemolysis (green discolouration)

- Further incubation, colonies become flat with raised edges and central umbonation -Concentric rings on surface when viewed from above - Draughtsman or carrom coin appearance 

- Large Mucoid clonies ( types 3 and 7)- abundant capsular material 

-Under anaerobic conditions, ß haemolysis in due to oxygen labile Hemolysin O

-Glucose broth – growth occurs as uniform turbidity 

- Readily undergoes autolysis due to the activity of intracellular enzymes 

-Autolysis is enhanced by bile salts, Sodium lauryl sulphate and surface active agents 

ANTIGENIC PROPERTIES

Important antigens

• ·         Capsule
• ·         Nucleoprotein antigen
• ·         ‘C’ carbohydrate antigen u C – reactive protein (CRP)

Type specific Capsular polysaccharide –diffuses into culture medium/exudates/tissues- Specific Soluble Substance (SSS)

Based on Antigenic nature of Capsular polysaccharide, pneumococci were classified into pneumonia causing  I, II, III and heterogenous IV group

IV group has 90 serotypes named 1, 2, 3 and so on

Serotyping carried out by agglutination of cocci with type specific antiserum, Precipitation of SSS with specific serum or capsule swelling reaction/Quellung reaction  demonstrated by Neufeld

Quellung reaction  (Neufeld in 1902)

Suspension of S. pneumoniae is mixed on a slide with a drop of type specific antiserum and a loopful of methylene blue - Capsule becomes swollen and refractile (scatter light)

Done directly with sputum in acute pneumonia cases- was done routinely in the past when the specific antiserum was used for treatment of pneumonia

CRP –Abnormal protein (beta globulin) which precipitates with the somatic C antigen of pneumococci-appears in acute phase of pneumonia, disappear later.

C reactive protein- common in other pathological conditions too.

Not an antibody produced in pneumococcal infections- produced in hepatocytes- stimulated by bacterial infections, inflammation, tissue destruction

Smooth to rough (S – R) variation

In the R form, cocci are non capsulated, auto agglutinable and avirulent –arise as spontaneous mutants in cultures and outgrow S forms, In tissues R mutants are eliminated by phagocytosis

Rough S. pneumoniae of one serotype may be made to produce capsules of the same or different serotype, on treatment with DNA from the respective serotypes

Transformation by Griffith (1928)- demonstration of genetic material in bacteria

 Biochemical Reactions

• ·         Catalase and oxidase negative
• ·         Ferments sugars with acid production -Fermentation tested in Hiss’s serum water **
• ·         Ferments Inulin-streptococci does not  
• ·         Pneumococci are bile soluble-of diagnostic importance
• ·      Bile solubility test: If a few drops of 10% sodium deoxycholate are added to 1 mL of an overnight broth culture, the culture clears due to lysis of cocci- due to an autolytic amidase that cleaves the bond between alanine and muramic acid in the peptidoglycan; Amidase is activated by bile salts

          

**(Hiss's serum water is used in carrying out fermentation tests with certain pathogenic organisms, such as the pneumococcus that do not grow well in the peptone–water solutions. Fermentation is indicated by the change in the indicator by the production of acid that may also coagulate the serum. Litmus milk is used for biochemical tests. Acid production, a result of the fermentation of lactose, turns the medium pink and may also coagulate it; proteolytic activity is indicated by digestion and alkali formation.)

Resistance

• ·         Pneumococci are delicate organisms.
• ·         Readily destroyed by heat- 52° C for 15 minutes and antiseptics
•    Cultures die on prolonged incubation due to accumulation of toxic peroxides- maintained by lyophilisation
• ·         Sensitive to beta lactam antibiotics initially- now resistant-resistance due to alteration in penicillin binding proteins on the bacterial surface
• ·         Pneumococci sensitive to Optochin (Ethyl hydrocuprein) sensitive- Streptococci are not

Toxins and Virulence Factors

• ·         Virulence depends on its capsule and the production of a toxin called pneumolysin
• ·         Capsular Polysaccharide (acidic & hydrophilic) - protects the cocci from phagocytosis
• ·      Enhanced Virulence of type 3 due to abundant capsular material- Non capsulated strains are avirulent - Antibody to capsular polysaccharide is protective
• ·         Pneumolysin –membrane damaging toxin produced by Pneumococci- cytotoxic and complement activating properties- virulence factor-immunogenic
• (Pneumolysin-negative mutants showed less virulence in experimental animals)
• ·         Pneumococcal Autolysins- release bacterial components in infected tissues- contribute to virulence
•  Oxygen labile hemolysin and leucocidin – not significant in virulence

Epidemiology

• ·         S. pneumoniae occurs in the throats of 50% of human population, any time 
•        Source of infection is the respiratory tract of carriers, and at times, of patients.
• ·         Transmitted by fingers or by inhalation of droplets
• ·         Dissemination is facilitated by crowding
• ·         Infection usually leads to pharyngeal carriage-- disease results when host resistance is lowered by viral infection, pulmonary congestion, stress, malnutrition, immunodeficiency or alcoholism

  Pathogenicity

• ·         Experimental infection in mice & rabbits by intraperitoneal inoculation - fatal infection – pneumococci demonstrable in peritoneal exudate and heart blood–death in 1-3 days

• ·         Colonise the human Nasopharynx – cause infection of middle ear, paranasal sinuses and respiratory tract – direct infection
• ·         Can spread through blood and lymphatic system– cause Meningitis – distant infections in the heart, peritoneum or joints
• ·         Infection is generally, endogenous in nature, can be Exogenous also

Common Pneumococcal infections 

• otitis media and sinusitis – prior respiratory infection or allergy can lead to these conditions

• common cause of lobar and bronchopneumonia –also cause acute tracheobronchitis and empyema (collection of pus in the pleural cavity)

•  Normal mucosal defense mechanisms (entrapment, cough, ciliary defense) prevent establishment of infection. But if these defense mechanisms are compromised by infections, anesthesia or chilling etc, Pneumococci can multiply and spread through lung and lymphatics- Bacteremia is common in lobar pneumonia

• Diffusion of capsular polysaccharide into the blood and tissues- cause toxemia.
•  SSS neutralized by anti-capsular antibodies produced by the patient- cause fall in temperature and general relief of symptoms
•  In adults, 50% fatalities are due to Pneumococcal bacteremia.

• Bronchopneumonia is always a secondary infection – damage to the respiratory epithelium and excessive bronchial secretions caused by the primary infection, lead to the invasion of Pneumococci to the bronchial tree- terminal event in aged and debilitated patients 

• Meningitis – most serious Pneumococcal infection
• Is a secondary infection after the primary infections like  pneumonia, otitis media, sinusitis or conjunctivitis - Occur at all ages 
• If untreated, highly fatal 
• 25% fatality even with antibiotic therapy

•  Pneumocci also cause suppurative lesions in different parts of the body- empyema, pericarditis, otitis media, sinusitis, conjunctivitis, suppurative arthritis, peritonitis , keratitis, dacryocystitis (lacrymal sacs infection)

Laboratory Diagnosis

• Clinical diagnosis of Pneumonia is easy -etiological diagnosis needs laboratory assistance- many organisms involved
• Specimen – Sputum, CSF, Blood and Urine
• Microscopy - In acute lobar pneumonia, rusty sputum contain Pneumococci in large numbers -hardly any other bacterium- Gram stain.

(Rusty sputum- sputum produced in pneumonia caused by S pneumoniae - contain bacteria, hemorrhage, mucus,  necrotic lung tissue.)

• In the pre-antibiotic era, direct serotyping with Quellung reaction followed by treatment with specific antiserum-routine process

• Culture – The sputum after homogenization, inoculated onto blood agar plates, incubated at 37^oC, under 5-10 % CO₂, overnight. In infants, laryngeal swabs may be used for culture. Gentamycin can be added to blood agar to facilitate isolation of Pneumococci.

• In acute pneumonia, the organism may be obtained from Blood culture in Glucose broth – isolation of pneumococci in blood indicates bad disease condition.

• Animal inoculation – Intraperitoneal inoculation in mice, if specimen contains scanty numbers of the organism and even if cultures are negative. Pneumococci may be demonstrated in the peritoneal exudates and heart blood. Inoculated mice die in 1-3 days.
• Test maybe negative if the strains are avirulent for mice (type 14 strain)

• In otitis media, Pneumococci demonstrated in fluid aspirated from the middle ear

• Meningitis- presumptive diagnosis from Gram stained films of CSF-Gram positive diplocoocci visible inside polymorphs and extracellularly. Diagnosis confirmed by culture. If negative in culture, SSS demonstrated in CSF by immunoprecipitation (with antisera)

• Antigen Detection  -SSS/Capsular polysaccharide -in blood, urine, CSF by Precipitation, counterimmunoelectrophoresis
• Antibody demonstrated by agglutination, precipitation, indirect haemagglutination, radioimmunoassay etc

Prophylaxis

• Immunity is type specific – antibodies against  capsular polysaccharide
• ` 90 serotypes- so complete polyvalent vaccine not practical
• A polyvalent polysaccharide vaccine with  capsular antigens of the 23 most prevalent serotypes gives 80 – 90 % protection 
• not meant for general use but for people at increased risk such as absent or dysfunctional spleen,  Sickle cell disease, chronic heart, renal, lung and celiac disease, Diabetes mellitus, HIV etc
• not recommended for children under two years of age and those with immunosuppressive therapies.

Treatment 

• Parenteral administration of Penicillin in serious cases and  Amoxycillin in mild cases (if the isolate is sensitive to penicillin)  
• Many Penicillin resistant (alteration in Penicillin Binding Protein)- strains are resistant to erythromycin and tetracycline
• For resistant isolates- Third generation cephalosporins and in life threatening illnesses with highly resistant strains, Vancomycin

Plant Viral Diseases

 

Tomato Yellow Leaf Curl Virus (TYLCV) is a major problem on tomato. Once the disease has occurred, it will spread fast and drastically reduce the yield.

Symptoms of infection yellowing of young leaves, upward and downward leaf curl , stunting, bushy appearance, and flower drop, which occurs prior to fruit set and dramatically reduces fruit yield.

Affected plants tend to be distributed in isolated patches. This virus can cause significant yield losses from 80-100%

Pathogen

TYLCV is of the genus Begomovirus and family Geminiviridae.

Leaf curl & Interveinal and marginal chlorosis on young tomato leaves infected with TYLCV

Host Plants

TYLCV predominately causes disease in tomatoes. It can infect other hosts in the Solanaceae family (pepper, eggplant, potato, tobacco, jimsonweed), as well as common bean (Phaseolis vulgaris L.), and ornamentals including petunia and lisianthus. In the absence of symptoms, these hosts may serve as a reservoir of the pathogen.

Signs and Symptoms

On tomatoes, the primary symptoms of TYLCV are interveinal and marginal chlorosis of young leaves, an overall crumpled appearance of the leaves, and upward and downward leaf-curling. Plants infected with TYLCV will also be stunted in height and appear bushy due to shortened internode length. Flower drop will occur with an accompanying reduction in yield. When young plants are infected, the disease may be so severe that no fruit is produced. Symptoms can take up to three weeks to develop after infection.

  

Look-alike conditions

Initial symptoms of TYLCV can resemble other disorders that affect tomatoes, including potassium deficiency, magnesium deficiency, micronutrient deficiencies, and leaf curl. In general, any of these nutrient deficiencies are likely to be consistent across the entire crop whereas plants infected with TYLCV will be patchy across the field and not uniform. This pattern may be used to help diagnose the problem. TYLCV differs from symptoms of nutrient deficiencies in that the marginal and interveinal chlorosis first appears on young leaves. Curling of new leaves may also be caused by excessive aphid feeding or other viruses. It is highly recommended to confirm the diagnosis by a plant disease clinic or extension specialist since there are several lookalike diseases and disorders.

Disease Cycle and Epidemiology

TYLCV is transmitted by adult whiteflies (Bemisia tabaci). A whitefly can acquire the virus within their salivary glands after 15-30 minutes of feeding on an infected plant. After a latent period (time between acquisition and transmission of virus) of ~6 hours, the whitefly is able to transmit the virus to another plant within 15-30 minutes of feeding. Adult whiteflies can retain and spread the virus for several weeks after initially feeding on infected plants and can travel over distances of at least 10 miles.

The virus has been detected on seed collected from infected plants. Yet, the virus survives only on the surface of the seed and is not transmissible to seedlings following seed-surface disinfestation. 

Following infection it may take up to three weeks for symptoms on tomato to appear due to the movement of symptomless infected plant material spreading the disease over long distances. A high population of white flies in mature stages of field-grown tomato production may serve as an inoculum source for younger plantings nearby.

Bemisia tabaci

 General Disease Management

• ·       Use disease resistant cultivars
• ·       Destroy old infested crop after harvest.
• ·       Raise barrier crops – cereals (3 rows of sorghum, cumbu and maize) around the field.
• ·       Remove weeds as these may harbour the virus.
• ·       Adopt polythene mulching technology during planting to control weed hosts.
• ·       Physically protect the nursery from the vector, e.g. net house or green house.
• ·       Keep yellow sticky traps to monitor the white fly.
• ·       Spray insecticides to control the vector.

Banana Bunchy Top

Banana bunchy top is a viral disease caused by a single-stranded DNA virus -banana bunchy top virus (BBTV), of the family Nanoviridae. It is aphid transmitted and infects banana plants and other crops. It was first identified in Fiji in 1879.

BBTV was named after the symptoms seen, where the infected plants are stunted and have "bunchy" leaves at the top. The disease is transmitted from plant-to-plant in tropical regions of the world by banana aphids, which is an important factor in control of the disease.

There are no resistant varieties, so controlling the spread by vectors and plant materials are the only management methods.

Host

Banana bunchy top disease affects the banana fruit and foliage. BBTV can infect species of the family Musaceae, which includes bananas, plantains, abaca, and more. The aphids also feed on Heliconia and flowering ginger.

Symptom

The pathogen causes cytopathological effects in the phloem tissue, due to damage of the host cells by the virus. The name of the disease is due to the symptom in older plants, in which the new leaves that are produced are narrower than normal, yellow, and flat, which causes a "bunchy" appearance at the top of the tree. 

If any fruit is produced, which is unusual, it will be deformed. One of the most distinctive symptoms is "Morse code streaking" causing irregular spots and dashes on the leaves which are easier to see when the waxy coating over the petiole is rubbed away. The infected cells die and are lighter in color hence this appearance.

Initially, dark green streaks appears in the veins of lower portion of the leaf midrib and the leaf stem. They appear to be “bunched” at the top of the plant, the symptom for which this disease is named. Severely infected banana plants usually will not fruit, but if fruit is produced, the banana hands and fingers are likely to be distorted and twisted. 

 

 

 Disease cycle

BBTV is the sole member of the genus Babuvirus in the family Nanoviridae.

It is known that Banana aphid (Pentalonia nigronervosa) transmits the virus from infected to healthy plants by feeding. Aphids feed on the plant phloem tissues by injecting their thin, flexible stylet into the epidermis of the plant tissue until it reaches the phloem of the leaves. Then the aphid injects saliva, sucks the cell contents and introduces virus in the process.

Vector transmission of the BBTV is non-propagative, and the virus does not replicate within the aphid’s midgut. Acquisition of the virus by the banana aphid requires about 18 hours of feeding and then the aphid can retain the virus for approximately two weeks. It takes about a month for the BBTV symptoms to appear after infection.

 To infect, the carrier aphid can feed on the banana plant for as few as 15 minutes, but more often a couple hours, as the longer feeding time will increase the odds of transmission. The suckers produced on infected plants will also be diseased thus the disease can spread from year to year.

Banana aphids also have the capability to feed on Heliconia and flowering ginger; however, these alternate hosts of the aphid vector are not hosts of the virus. The ability of banana aphids to feed on alternate hosts is important to keep in mind when attempting to control the virus.

 Management

• There are no resistant varieties of banana against BBTV, so the most common method of control is chemical control of the aphid vectors.
• Another way to help control the virus is to remove and destroy any infected plants before the virus can spread, which is a practice known as roguing.
•  Quarantines are also implemented to prevent the import of any potentially infected plant materials
• Fruit is not often produced on infected plants, but if it is, the fruit will be deformed, which easily identifies if there is any virus present in the fruits to comply with quarantine regulations.
• Since bananas are not the only host, the alternate hosts for both the virus and the aphid must also be monitored for disease, and sprayed with pesticides to control the aphids more.
• When planting at the beginning of the season, the seed material or suckers should be obtained from BBTV free areas of the world or from cultures that are grown and developed to be free of the virus.
• Control of banana bunchy top is achieved by killing the banana aphids then destroying all infected material. First, the aphids should be killed on the infected banana material, and then all the plant material should be destroyed to prevent the spread of the virus. 

Importance

Banana bunchy top disease is the most serious virus disease of banana worldwide. Diseased plants rarely produce fruit and when they do, the fruit is stunted and twisted.

BBTV certainly has a huge impact on the industrial scale of banana production and affects the livelihood of farmers.

Once established, it is very difficult to eradicate and manage the disease. 

First of all, the disease is caused by a vector-transmitted virus and this virus is not completely understood yet. Secondly, all bananas are susceptible to the disease and no resistant varieties have been discovered or made commercially available. Lastly, the control methods are quite demanding, including chemical treatment for the aphid vectors, removal of all infected tissue, quarantining plants and monitoring alternate vector feeding sites.

Epistasis, Pleiotropy

 EPISTASIS 

In epistasis, the interaction between two genes is antagonistic, such that one gene masks or interferes with the expression of another. “Epistasis” is a word composed of Greek roots that mean “standing upon.” The alleles that are being masked or silenced are said to be hypostatic to the epistatic alleles that are doing the masking. Often the biochemical basis of epistasis is a gene pathway in which the expression of one gene is dependent on the function of a gene that precedes or follows it in the pathway.

 The phenomena where the effect of one gene depends on the presence of one or more gene, is known as epistasis. The phenotypic effect of one gene is masked by another gene. The gene which masks the effect of another gene is known as epistatic gene. The epistatic genes can be dominant or recessive in their effects and the gene whose effect is masked by the epistatic gene is known as hypostatic gene.

By the definition, confusion arises between the dominance and epistasis but dominance involves intra-allelic gene interaction and one allele hides the effect of other allele at the same gene pair, whereas in epistasis it involves inter-allelic gene interaction i.e. one gene hides the effect of other gene at other gene loci.

For the study of linkage association analysis, epistasis plays an important role. Epistatic mutations therefore have different effects on their own than when they occur together. Epistasis has a great influence on the evolvability of phenotypic traits.

 

TYPES OF EPISTASIS

 Dominant Epistasis When a dominant allele hides the effect of allele of another gene and expresses and itself phenotypically, is known as the dominant epistasis. The hypostatic allele will only get expressed when the gene locus contains two recessive alleles. The expression of one dominant or recessive allele is masked by another dominant gene. This is also referred to as simple epistasis.

An example of dominant epistasis is found for fruit colour in summer squash. There are three types of fruit colors - white, yellow and green. White colour is controlled by dominant gene W and yellow colour by dominant gene G. White is dominant over both yellow and green. The green fruits are produced in recessive condition (wwgg). 

A cross between plants having white and yellow fruits produced F1 with white fruits. Intermating of F1 plants produced plants with white, yellow and green coloured fruits in F2 in 12:3:1 ratio.

FIG- The figure explains the dominant epistasis for fruit colour in summer squash. The normal dihybrid modified to12:3:1 in F2 generation. Here W is dominant to w and epistatic to alleles G and g. Hence it will mask the expression of G/g alleles. Hence in F2, plants with W-G-(9/16) and W-gg (3/16) genotypes will produce white fruits; plants with wwG-(3/16) will produce yellow fruits and those with wwgg (1/16) genotype will produce green fruits. Thus the normal dihybrid ratio 9:3:3:1 is modified to 12:3: 1 ratio in F2 generation.

Similar type of gene interaction has been reported for skin color in mice and seed coat color in barley.

 Recessive Epistasis

The recessive allele of one gene locus hides the effect of another gene locus and expresses itself phenotypically. When recessive alleles at one locus mask the expression of both (dominant and recessive) alleles at another locus, it is known as recessive epistasis.

An example of recessive epistasis is pigmentation in mice. The wild-type coat color, agouti (AA), is dominant to solid-colored fur (aa). However, a separate gene (C) is necessary for pigment production. A mouse with a recessive c allele at this locus is unable to produce pigment and is albino regardless of the allele present at locus A (Figure 1). Therefore, the genotypes AAcc, Aacc, and aacc all produce the same albino phenotype. A cross between heterozygotes for both genes (AaCc x AaCc) would generate offspring with a phenotypic ratio of 9 agouti:3 solid color:4 albino. 

In this case, the c gene is epistatic to the A gene.

 

 

A-C-  agouti          A-C- Black                A-cc white

Another good example of such gene interaction is found for grain colour in maize. There are three colours of grain in maize, viz., purple, red and white. The purple colour develops in the presence of two dominant genes (R and P), red colour in the presence of a dominant gene R, and white in homozygous recessive condition (rrpp). A cross between purple (RRPP) and white (rrpp) grain colour strains of maize produced plants with purple colour in F1. Inter-mating of these F1 plants produced progeny with purple, red and white grains in F2 in the ratio of 9:3:4.

 Recessive epistasis for grain colour in maize.    RRPP x rrpp

The normal dihybrid segregation ratio 9:3:3:1 is modified to 9:3:4 in F2 generation. Here allele r is recessive to R, but epistatic to alleles P and p. In F2, all plants with R-P-(9/16) will have purple grains and those with R-pp genotypes (3/16) have red grain color. The epistatic allele r in homozygous condition will produce plants with white grains from rrP-(3/16) and rrpp (1/16) genotypes. Thus, the normal segregation ratio of 9:3:3:1 is modified to 9:3:4 in F2 generation.

 Such type of gene interaction is also found for coat color in mice, bulb color in onion and for certain characters in many other organisms.

 

Pleiotropism

 Pleiotropism is the condition in which a single gene controls more than one phenotypic effect, that is completely unrelated.  Pleiotropy is a condition in which a single gene has multiple phenotypic expressions.

E.g.: Phenylketonuria It is an autosomal recessive disorder due to problem in chromosome number 12 

When the phenylalanine levels are affected, it causes a disease known as phenylketonuria- due to the defect in single gene present on chromosome 12 which codes for the phenylalanine hydroxylase. Phenylalanine is an essential amino acid obtained from food and causes multiple effects such as mental retardation, hypopigmentation of hair and skin.

 

 Other examples of the pleiotropy are albinism, sickle cell anemia, autism, etc. Pleitropy not only affects humans but also its affect is seen in the animals as well like, chicken, mice, etc.

• Sickle cell disease is caused by a problem in the hemoglobin-beta gene found on chromosome 11. The defect forms abnormal hemoglobin.