eChapters

A. Bacterial Diseases in Shrimp

Bacterial diseases in hatcheries and farms are being increasingly recognised as major hurdles to successful shrimp farming (Plate 6). Among the several bacterial diseases, diseases due to Vibrio sp. (vibriosis) are common. As majority of the Vibrio are secondary opportunistic pathogens, problems related to vibriosis can be traced to stress, poor water quality and bad management. It is widely accepted that the ubiquitous opportunistic vibrios are always responsible for causing mortalities in shrimp which are already stressed by poor water quality, external fouling and primary pathogens like viruses.

Vibrios are gram negative, oxidase positive, motile rods. Most frequently reported species in hatchery situations include Vibrio harveyi, V. vulnificus, V. parahaemolyticus and V. alginolyticus. All species of cultured shrimp are susceptible to vibriosis under stressful condition.

The common clinical signs associated with vibriosis are: 1) high mortalities, particularly in PL and young juveniles shrimp, 2) Moribund shrimp appearing hypoxic and often coming to pond surface and edges, 3) Reddening of shrimp, 4) Shell and appendage necrosis with blackening, 5) Presence of luminiscence in affected shrimp in tanks and ponds.

Separation of Bacteria and Protozoa
(Method of Czerkawski, 1975)

Rumen contents are obtained just before and two hours after feeding from sheep that had been given an experimental ration. The samples of rumen contents are strained through gauze and fractioned by sedimentation and centrifugation to give preparations of mixed protozoa (PR), large bacteria (LB) and small bacteria (SB). Whole particulate matter is also isolated by centrifugation at 20000g. The detail steps are mentioned in the flow chart given below.

Introduction

The soil around plant roots that forms the rhizosphere is a dynamic, complex zone. All plant parasitic nematodes are obligate parasities and must enter this zone to reach their host and cause damage (Kerry, 2000). Nematodes in soil are subject to infections by bacteria and fungi. This creates the possibility of using soil microorganisms to control plant-parasitic nematodes. Bacteria are numerically the most abundant organisms in soil, and some of them, for example members of the genera Pasteuria, Pseudomonas and Bacillus have shown great potential for the biological control of nematodes. Nematophagous bacteria are distributed broadly, possess diverse modes of action, and have broad host ranges. A variety of nematophagous bacterial groups have been isolated from soil, host-plant tissues, and nematodes and their eggs and cysts (Siddiqui and Mahmood, 1999; Kerry, 2000; Meyer, 2003).

They affect nematodes by a variety of modes: for example parasitizing; producing toxins, antibiotics, or enzymes; interfering with nematode–plant-host recognition; competing for nutrients; inducing systemic resistance of plants; and promoting plant health (Siddiqui and Mahmood, 1999). They form a network with complex interactions among bacteria, nematodes, plants and the environment to control populations of plant-parasitic nematodes in natural conditions (Kerry, 2000).

Bacterial culture methods are key to diagnosing and confirming bacterial infections in fish. These techniques involve the isolation and growth of bacterial pathogens from fish tissues or environmental sources under controlled lab conditions. The effectiveness of these methods depends greatly on accurate sample collection, careful handling, and the correct choice of culture media and incubation parameters.
Samples are aseptically collected from infected fish—typically targeting tissues such as the kidney, liver, spleen, gills, and any visible skin lesions. In live fish, sterile tools are used for tissue extraction, while in dead fish, internal organs should be harvested promptly after death to reduce contamination. For surface-level infections, swabs taken from mucus or skin lesions are also helpful. To maintain bacterial viability and minimize contamination, samples must be kept cool during transport.
In the laboratory, samples are inoculated onto culture media. The type of media used depends on the suspected pathogen. Common general-purpose media include Tryptic Soy Agar (TSA), Nutrient Agar (NA), and Brain Heart Infusion Agar (BHIA). For selective isolation, media such as MacConkey Agar (for Gramnegative bacteria), TCBS Agar (for Vibrio species), or Edwardsiella Isolation Medium are utilized. Enrichment broths like Alkaline Peptone Water or Buffered Peptone Water may be used to boost bacterial numbers before plating.
Incubation typically occurs at 25°C to 30°C, with most fish pathogens growing well in this range. Plates are monitored over 24 to 72 hours. Bacterial colonies are assessed for physical traits such as size, shape, color, and hemolysis patterns, particularly on blood agar.

Wild life diseases are given more priority in the recent days due to the fact that conservation of any wild fauna canít be achieved fully, unless due efforts are equally given pertaining to the assessment of health status and the disease management, in the concerned wild animal species.

Ducks are members of the order Anseriformes and belong to the family Anatidae. Ducks, swans and geese are all members of the Anatidae family; however, ducks are smaller and have a shorter neck. Ducks are tough birds that require little care or management. People all over the world enjoyed and consume duck meat and eggs. Ducks produce more eggs than hens. The size is also larger than that of a chicken. Ducks require less attention and perform well in scavenger hunts. Ducks are second only to chickens in many countries for meat and eggs. Ducks can be raised commercially or on a small scale for meat or eggs, even in backyards with other birds or animals. 

With increasing fish culture activities several bacterial diseases causing morbidity and mortality in fish have been reported in tropical and subtropical aquaculture systems. Common and major diseases are due to motile aeromonads, Vibrios and Pseudomonas. However, bacterial diseases of minor nature have also been reported from aquacultural systems. In the last two decades several new diseases due to Streptococcus sp, Edwardsiella and Ricketsia have also emerged.

Bacteria can cause disease either as primary pathogens or as secondary opportunistic invaders. Bacterial diseases in fish can be broadly classified as surface ulcerative, acute systemic and chronic granulomatous types (Table 1, Plates 4 and 5). Surface ulcerative types of diseases are characterized by hemorrhagic surface ulcers and are caused by species of Aeromonas, Pseudomonas, Vibrios, Flexibacteria and Myxobacteria. Surface ulcerative disease conditions at times develop to acute systemic disease. Acute systemic disease are characterized by the presence and proliferation of bacteria in internal organs like kidney, heart, spleen, blood and other visceral organs. These diseases produce significant necrotic changes in all affected organs and can cause mortality in a short time scale. Bacterial hemorrhagic septicemia caused by Aeromonas hydrophila is a major problem in carp farms. Chronic granulomatous type of disease conditions are characterized by the formation of granulomas or nodules with the initiating bacteria in the centre. These conditions will not produce mass mortality but can produce chronic low levels of mortality and can significantly reduce growth.

Introduction Shrimp farming is a lucrative sector in the food production industry. According to SOFIA (2020), 9.4 million tonnes of crustaceans were produced in 2020 worldwide, with 4.96 million tonnes coming from Penaeus vannamei. India is one of the largest global producer and exporter of farmed shrimp. In India, shrimp production increased significantly after introducing Pacific white leg shrimp, Penaeus vannamei, in 2009. Among the cultured shrimp species, Penaeus vannamei is the leading species under culture in India, with an estimated production of 7.12 lakh tonnes (MPEDA, 2020). Intensification of culture and diversification of species provoke the incidence of diseases making diseases the major constraint in aquaculture, leading to tremendous losses. Shrimp diseases are of viral, bacterial, fungal and parasitic origin.

Clinical Symptoms

• Gasping for air

• Discoloured gills with trapped materials

• Swollen gills with enhanced mucous secretion

• White to grey spots on the gills

• Diseased fish gather near the outlet of the pond

• Fish become lethargic and remain on the surface waters

Causative Agent: Flavobacterium branchiophilum, Cytophaga spp., Flexibacter spp.

Host (Common sp.)

• Goldfish • Koi carp

• Cichlids • Fighter fish

• Gourami fish

1. How does the toxin of Clostridium botulinum affect the nervous system?

a) Blocks inhibitory transmitter substance

b) Stimulates macrophages

c) Causes haemolysis and tissue destruction

d) Induces systemic shock and death

What is the specific effect of the toxin produced by Clostridium perfringens on host cells?

a) Blocks inhibitory transmitter substance

b) Induces systemic shock

c) Causes ribosomal dysfunction

d) Disrupts plasma membranes

1 Which of the following diseases is also known as Equine Distemper?

a) Eastern Equine Encephalomyelitis

b) Western Equine Encephalomyelitis

c) Strangles

d) Glanders

Introduction

Microorganisms are a great source of genetic diversity, still far from completely known and explored. The intimate association of plants with complex microbial communities is of great importance for the growth and productivity of plants in agriculture. There exists a definite difference in microbial community present in the bulk soil and the neighboring micro environments like phyllosphere, endosphere, rhizosphere and endorhizae. Microorganisms that colonize the plants’ rhizosphere are either pathogenic or beneficial according to their effects on plants and the way they interact with roots. The rhizosphere supports a plethora of microbial populations capable of exerting beneficial, neutral or detrimental effects on plant growth and development. Microorganisms such as bacteria, actinomycetes, protozoa, algae, fungi, and viruses coexist in the rhizosphere; bacteria are the most abundant among them.

The genetics is the study of inheritance and variability of organisms. Bacterial genetics is a sub-field which specifically studies transmission of genetic information from one bacterium to its progeny or between the interbreeding bacteria and the expression of genetic information.

Mutation

Hugo de Vries (1848-1935, Dutch botanist) coined the term ‘mutation’. It is originated from a Latin word which means ‘to change’. The heritable change in bacterial DNA sequences is known as bacterial mutation. The mutation will generate a new type of bacteria with altered genotypical and phenotypical characteristics, known as ‘mutant’. The parent bacterial cell from which the mutants are generated is known as ‘wild type’. In nature bacterial mutation is a rare incidence. The spontaneous mutation occurs at a rate of 1 in 10⁵ to 10⁸. The phenotypical expression of mutation occurs quickly as the bacteria are haploid with shorter generation time.

What are Genes? Genes are linear sequences of nucleotides of DNA (deoxyribonucleic acid). Each gene consists of a specific sequence of nucleotides and is responsible for determining a particular characteristic of cell. Nucleotides consist of nitrogen –containing compounds called bases, joined to the sugar, deoxyribose and to phosphate. The nucleotide bases are the purines, adenine and guanine and the pyrimidines, thymine and cytosine.

A number of genes are joined together to form a structure called a chromosome.

DNA consists of two strands of nucleotides twisted around each other into a double helical structure. The nucleotide bases are arranged in a specific sequence in each strand. The strand has a backbone of sugar and phosphate from which the bases protrude.

Etiology

The condition is usually caused by implantation of low virulent saprophytic bacteria due to traumatic injury. These organisms stimulate cell mediated immune response by persistence of antigen in the tissue.

10.1 Transformation of E. coli by electroporation Principle

Preparation of electro-competent bacteria is considerably easier than preparing cells for transformation by chemical methods. Bacteria are simply grown to mid –log phase, chilled, centrifuged, washed extensively with ice cold buffer containing 10% glycerol. DNA may be introduced immediately into the bacteria by exposing them to a short high voltage electrical discharge. Alternatively, the cell suspension may be snap frozen and stored at-70^oC for 6 months before electroporation, without loss of transforming efficiency.

Since E.coli cells are small, they require very high strengths (12.5- 15KV cm^-1) for electroporation as compared to those used to introduce DNA into eukaryotic cells. Electroporation is temperature dependent and is best carried out at 0-4°C.

The highest efficiency of transformation is obtained when the concentration of input DNA is high (1-10 μg/ml) and when the length and intensity of the electrical pulse are such that only 30-50% of the cells survive in the procedure. Under these conditions, as many as 80% of the surviving cells may be transformed. Higher frequencies of transformation (colonies/ molecules input DNA) are obtained when the DNA concentration is low (?10pg/ml).

The method outlined in this protocol is followed with most strains of E.coli and with plasmids <15kb in size. However, substantial variation in the efficiency of transformation between the strains of E. coli. has been reported. As in case with chemical transformation, linear plasmid DNAs introduced into E.coli by a pulsed electrical discharge transform very efficiently from 10 to 1000 fold but less efficiently than the corresponding closed circular DNA – perhaps because the exposed terminal of linear DNA are susceptible to attack by intracellular nucleases.

To Prepare the Competent Cell and Carry Out Transformation Introduction

Transformation results from the uptake of purified DNA by bacterial cells and is the most frequently used procedure for introducing recombinant DNA molecules into bacteria. It is now widely used to transfer small plasmids from one bacterial strain to another. DNA is extracted from donor cell and used to treat the recipient cells that have been rendered more susceptible to DNA uptake. These competent cells allow DNA to enter through pores or channels in the cell membrane and in the case of plasmids, permit subsequent plasmid replication.

Introduction

Bacteriological examination of milk indicates the hygienic condition of the milk i.e., quality of milk in relation to the health of consumer. Two main objectives of carrying out bacteriological examination are to evaluate the degree of cleanliness in the vicinity of production and handling of milk; and secondly to check the presence of infectious microorganism. Generally, number of bacteria present per milliliter is taken as an index in the routine examination of milk as other tests for identification and differentiation of pathogenic and non-pathogenic microorganisms are time consuming and expensive.

Direct Microscopic Count (DMC) consists of examining stained films of measured volume of milk or milk product, dried on a glass slide under a compound microscope. This method enables rapid estimation of total population of bacteria of a milk sample with relatively more accuracy as compared to other methods.

Cyanobacterial species

Cyanobacterial species viz., Anabaena variabilis, Anabaena fertilissima, Nostoc muscorum, N. punctiforme, N. linckia, N. commune, Spirulina platensis, Westiellopsis prolifica and Hapalosiphon sp. were obtained from National Center for Conservation and Utilization of Blue-green Algae, Indian Agricultural Research Institute, New Delhi. Cultures were maintained in BG-11 growth medium throughout the study in the laboratory.