Etiopathogenesis of hereditary and parasitic diseases

WORKER GUT

The guts of honeybee (Apis mellifera) adult workers are dominated by a distinctive set of nine

bacterial species (or ‘phylotypes’), largely

restricted to the hindgut.

Five of these, Snodgrassella alvi, Gilliamella

apicola, two species of Lactobacillus, and a

Bifidobacterium species, are ubiquitous and

can be found in every adult worker worldwide;

these species can be considered as the core gut

microbiome.

Others (Bartonella apis, Apibacter adventoris,

Frischella perrara, and Acetobacteraceae) are

present in guts of many honeybee workers, but

sometimes absent. The mature worker hindgut

8 9

microbiome is substantial, totaling 10 –10

bacterial cells and is established in workers within

four days following eclosure, before leaving the

hive.

Transmission is through a faecal route and

facilitated by social interactions and contact with hive surfaces. Each core bacterial species shows

a characteristic distribution within the hindgut. S. alvi and G. apicola dominate the ileum region of

the hindgut, where S. alvi forms a continuous layer on the lining of the longitudinal folds, and G.

Pagina 10 di 52

apicola occurs on top. F. perrara forms a melanized scab at the pylorus, near the beginning of the

ileum.

The others, which are Gram positive species, are most abundant in the rectum region of the

hindgut.

Parasites, symbiotic ones, as in mammals, have two functions:

1. Metabolise food.

2. Defence.

In fact, they are important to prevent the infection by competing the nutrition molecules with

pathogens (also for colonisation of mucosa side).

OVERVIEW OF ROLES OF THE GUT - MICROBIOME IN HONEYBEE HEALTH

In animals, gut microbiome composition is influenced by many factors, including diet, stress,

immune responses, aging, and exposure to antibiotics. All these factors appear to affect the bee

microbiome. Some evidence suggests that, as workers age and transition to foraging, the

microbiome composition

shifts slightly.

Microbiome composition,

particularly the relative

numbers of S. alvi and G.

apicola, can also shift through

the season, possibly

reflecting changes in diet.

Indeed, poor nutrition has

been shown to disrupt the

normal gut microbiome,

resulting in higher mortality

and disease susceptibility.

Disruption of the microbiome

(dysbiosis) has many

consequences for worker development: such disruption during early adult life affects expression of

important developmental genes and is expected to affect immune system function, since the

honeybee microbiome stimulates immune pathways. In turn, honeybee innate immune function has

been shown to be compromised by stimulation of cellular stress responses. Together these

findings suggest that dysbiosis may have cascading effects for the ability of bees to respond to

environmental stressors such as poor nutrition or temperature stress, and that, conversely, these

stressors may impact the microbiome.

Extremely specific parasites are present in the medium part of the bees’ gut, such as Gillianella or

Frischella. These types of bacteria are different from Lactobacillus or Bifidobacteria of mammals;

this because the conditions of the arthropod gut are very different from mammals, this causing the

colonisation from other microorganisms (they have some Bifidobacteria etc. but only in the

extremities, where there is also a major probability to have a recolonisation).

These bacteria can affect:

- Immune function. Pagina 11 di 52

- Metabolism.

- Present the ability of pathogens to develop on the mucosa – colonisation.

- Eliminate toxins.

- Lack of food and water = but this lack can cause problems not only to pathogens but also to

the permanent flora, this causing their destabilisation and consequently the growth of

dangerous bacteria.

Microplastics = they are made of hard material that can damage the epithelium of honeybees,

especially of the mucosa. They can also have some biochemical events such as the lack of the

expression of mediators of the honeybees.

But probiotics may be used to rebalance the good flora, even if this permanent flora is different

from mammals. Because of this, in fact, it’s necessary to use the correct mix, to help the

colonisation by the permanent flora (they can implement or be an alternative to pharmaceuticals).

First leg = immune response. Second leg = social interaction. Third leg = probiotics

Ppt 2

LEISHMANIASIS: IMMUNOPATHOLOGY AND ANIMAL MODELS

In the world there are different emerging parasites and vectors. Some examples are sand flies,

mosquitoes, ticks, and fleas. These may cause the transmission of diseases called zoonoses.

Environment is particularly important. It is influenced by different factors such as globalisation, land

use, climate change and animal travels (can spread the disease, the pathogens, because in the

new environment there is already the presence of potential vectors).

LEISHMANIA IN DOGS

Leishmaniosis is caused by a Pathogen, a protozoon, which can also affect humans (at least 22

classified species, among these 18 species that cause diseases in humans).

There are differences based on the geographic distribution (Old/ New World), of the caused

disease and of the vertebrate-invertebrate hosts (Isoenzymatic and molecular typing).

[Protozoa, class: Zoomastigophora, order: Kinetoplastida (Kinetoplast: extranuclear DNA), family:

Trypanosomatidae]

They are “Dixeni”: trasmission from haematofagous invertebrate to vertebrate and vice-versa.

The most dangerous can affect the “viscere,” the internal organs. But the majority causes problems

only to the skin. Pagina 12 di 52

The vector of Leishmania is a diptera, Phlebotomus, such the sand flies.

In Italy: P. perniciosus, P. perfiliewi, P. neglectus

Cycle of 4-20 days, 2-3 mm of length.

Sand fly has small hairs on the surface of the wings, very long legs, and

the body is L-shaped.

The difference between male and female in Leishmania is link to the

sexual structure, which is implicate in the reproduction. This structure is present only in female, the

important subject for the transmission of the disease: indeed, only females are hematophagous.

While in the dog the principal Leishmania is Leishmania infantuum, in humans there are two main

species, L. donovani and L. infantum. In humans, however, Leishmania can cause problems and

death, even if the majority causes skin problems.

The World Health Organisation estimates that leishmaniasis is endemic in 88 Countries throughout

the world.

Biological Cycle

In temperate climates and mediterranean regions the flies are more present from May to October,

while in the winter they are not able to survive.

The development cycle starts first in stomach/midgut and then spreads in pharynx/foregut

(suprapylaria). Pagina 13 di 52

Their hosts are especially mammals, but some flies can beat also lizards (reptiles), to assume the

survival of the vectors.

There are some studies on the role as a vector of the ticks (zecche). In fact, we can find

Leishmania in them, but we cannot say that they can transmit it. There is no epidemiological

evidence.

Cycle:

1) Promastigote = it has a flagellum, which can help the pathogen moving from the gut,

where it enters, to the salivary glands, where it can be transmitted. The salivary is

important because it is immunomodulatory and vasodilatator. It is transmitted during the

bloodmeal.

2) Amastigote = without flagellum, it is the endocellular stage. This lack is because it is

not useful anymore. It is phagocytised from monocytes, but not dissolved. In this way it

is carried in the body.

The exchange of blood materials can be method of transmission of Leishmania, especially

from dogs, where, even if infrequent, a possible vertical and venereal transmission can occur.

Non-conventional transmission ways: it happens only when there is no other way, no other vectors

(the dog in fact is the obligate winter reservoir).

There is the possibility of alternative reservoirs when:

1. ↑ in humans but no ↑ in dogs

2. human strain(s)≠canine strain(s). →analyse of vector tropism

For this reason, it is important to detect new areas to detect new endemic focus. We have to

identify autochthonous cases (ill dogs symptoms, analyse the positivity and do an anamnesis), it is

important to do a serological monitoring of residence dog (such as with ELISA, we can find at least

2-3% of positive dogs) and finally do a entomological monitoring (understand how flies work, where

they live, their preference, know the species to understand the primary prey). Pagina 14 di 52

We can also have areas that are not fixed, because they are linked to sporadic conditions (ex, an

abnormal hot winter), that have next year disappeared. But unfortunately, these cases are rare.

Normally are permanent.

Foci of visceral leishmaniasis: predisposing factors

• Movement of infected dogs from endemic toward non-endemic areas with presence of

vectors

• Progressive increase of distribution and/or density of vectors

• Insufficient monitoring of resident canine population

METHODOLOGY FOR THE STUDY OF NEW TRANSMISSION FOCI: strain identification from

autochthonous clinical cases

• Strain cultivation from biological samples (lymph node / bone marrow aspirates)

• Isoenzymatic/molecular typing of the isolate

• Non-attractive traps (castor oil) or Attractive lighting traps (CDC) or Manual catching devices

! Not all the flies are prone to be captured using light traps, because they have different types of

sensibility.

After capturing the flies is necessary to use Molecular strategies to study the sample.

A NESTED-PCR can be used: the first passage is very general, can help to recognise the

pathogens. The second one is more specific, to identify the precise pathogen (species or strains).

After that a sequencing can be performed.

Nested polymerase chain reaction (PCR) is used in situations in which it is necessary to increase

the sensitivity and/or specificity of PCR.

Nested PCR usually involves two sequential amplification reactions, each of which uses a different

pair of primers. The product of the first amplification reaction is used as the template for the second

PCR, which is primed by oligonucleotides that are placed internal to the first primer pair.

The use of two pairs of oligonucleotides allows a higher number of cycles to be performed, thereby

increasing the sensitivity of the PCR. The improved specificity of the reaction derives from the

binding of two separate sets of primers to the same target template. Nested PCR is an efficient

method to amplify segments of long templates but requires knowledge of the sequence of the

target. Pagina 15 di 52

Infection dynamic in dogs: clinical signs and immunological characteristics (L. infantum infection)

- Severe non-self-limiting disease = Th2 predominant response (high serology, decreased

cellular immunity and high parasite load causing the dissemination of the infection)

- Clinical