Leishmaniosi of Neapolitan Mastiff

HISTORY

MORPHOLOGY

The Leishmanie belong to the order of Kinetoplasti Tripanosomatide Family and has characterized from the presence of a little organ containing extra nuclear DNA said Kinetoplasto.  For the presence of a basal corpus joint to the Kinetoplasto we can observe three morphological types of Leishmanie: Amastigote, Promastigote and Paramastigote.  
Amastigote: it is the form that we observed in the vertebrate guest and in endocellular center and this shows a roundish shape contained inside of macrofagi.  Promastigote: it is the typical shape found in the vector insect.  It is characterized the presence of a long free tail.  Paramastigote: recent studies have individualized that during the cycle in the invertebrate guest, the Leishmanie present themselves in a more globular form with the tail that bathes in the citoplasma of the cell having an inflated appearance.

BIOLOGICAL CYCLE

The Leishmanie are organism’s dixeni that complete their vital cycle between two guests, an invertebrate that represents the vector, and a vertebrate that constitutes the reservoir of the illness.  The man has the role of occasional guest while the dog represents the main reservoir of Leishmanie.

CYCLE IN THE GUEST VECTOR

The cycle begins when a female of Phlebotomist feeds upon an infected vertebrate (dog, man, rat).  The ingested amastigoti are those freed from the cells during the suction and their number can also be very low.  The forms of amastigoti ingested with macrophage are released in the bowel of the vector insect where they start a transformation in promastigoti.  Here begins a transformation in promastigoti attached to the walls of the bowel of the phlebotomist, or there is a migration of the parasites with a shape of free promastigoti to the oral apparatus ready to be introduced in a vertebrate guest.  The cycle last from four to twenty days and could be influenced form the temperature: it is inhibited to temps of 10 degree C while it is accelerated to temperatures superior 20 degrees C.

CYCLE IN THE VERTEBRATE GUEST

One hypotheses of transmission of free promastigoti to the vertebrate guest are tied up the immission of the trunk in the skin and the penetration happens through the flow of saliva.  Once introduced the circulatory stream the cells are fagocitated by elements of the cells macrofagiche, to the inside of which the promastigoti divide for binary division, in the vacuole parassitofori until they reach such a number to break the macrofago, the freed amastigoti are fagocitated from the other macrofagi, repeating the cycle more times, In each case a part of them goes toward the skin and peripheric haematic circle become available to the repetition of the cycle.

THE VECTOR

Several hypotheses have been considered during the years before identifying the vector insect of the pathology.  The responsible has been identified in a bug of the Phlebotomist  kind.  The phlebotomists are bugs of small 2-3 mm of yellow pale or yellow rust color, the body and wings are covered by a dense down.

The female of phlebotomist require a meal of blood for the maturation of the eggs and the cycle foresees four larval forms and a pupale.  The development is influenced by the temperature and during the cold season, the larval forms could be in a phase of diapauses. 
During June to September period the larva’s are able to overcome till two generations of adult.  The phlebotomists are nighttime bugs that begin their activity at night fall with peaks of maximum activity around Midnight.  Their activity is disturbed by the meteorological conditions: lowering of temperature, wind and damp.

In Italy, the species of more diffused phlebotomists are the Perniciosus, the Perfiliewi and the Sergentomyna Minuta.

In Campania the most diffused species is the Perniciosus, while Perfiliewi has resulted almost completely absent and the Sergentomyna minuta is more diffused than the Perfiliewi.

In Tuscany is diffused the Phlebotomus Perfiliewi and the Sergentomyna minuta, while the Perniciosus is the less diffused.  (Maroli and other).

Other mammals have resulted positive of Leishmaniosis in other parts of the Mediterranean: the fox has resulted infected in Italy, France and Portugal, the black rat (Rattus rattus) has resulted positive in Italy, Jugoslavia and Spain.

The fox presents a chronic viscera-cutaneous infection similar to the dog.  The rat doesn’t present any symptom logy of the illness but this rodent is highly infectious for the phlebotomists vectors This shows that the pathology could be transmitted, also in absence of the canine guest, to the man and dog.

INTERACTION PARASITE GUEST AND PATHOGENESYS

An active role in the pathogenesis of the Leishmaniosi is constituted by the presented macrofago, that’s why it becomes important to individualize the mechanisms involved in the induction of the pathology.  Different structural factors, biochemical, immunological intervene either in the phase of initial bond or in the intercellular one of the parasite.

THE BOND LEISHMANIA-MACROFAGO

On the membrane of the parasite are present receptors of glicolipidic nature, two forms of glicoconiugati have been individualized: one cellular, tied up to the lipids of membrane, the other extracellular, constituted from hydrophile molecules which itself exclusively to the macrofagi and no to the other linfoid cells.  To the phase of binding (bond) follows the phagocitosys of the parasite that is englobed, at first in a fagosoma and therefore in the complex fagolisosoma.

The ability of survival of the Leishmaniia in the macrofago could be tied up to the oxidative activity of the fagocita: we have seen that the macrofagi parassitati, are able to kill and digest in vitro of the amastigoti only if continually stimulated by the linfochine (LK), substances that increase the phagocitative activity, lisosomiale and oxidative.  The production of linfochine is one of the points hinge of the immune answer, their deficiency of their could therefore be the base of the immunological dysfunction that is compared in case of Leishmaniosi.

PATHOGENESYS OF LEISHMANIOSI IN DOG

One of the possible cellular reactions to the entrance and to the persistence of the parasite in the susceptible cell is that of signal the immune system, through the exteriorization of the antigen leishmania, the infection in act. Therefore a resolution of the aggressive event from part of the immunocompetent system or a situation of quiescence could be.
In  the case of unfavorable eventuality, of organic subjugation of the guest, the parasite colonizes all the structures of the system phagocitosys mononuclear (SPM). Though being the macrofagi appointed to destroy them, they accept the plasmidi in their citoplasma. The slow permanence of the antigen parassitario with the immunocompetent cells is the base of the pathogenic evolution of the illness. The immunitary system of the organ parassitato blocks irrimediabyli, with a depth unbalance in the humoral answer from hyperfunzionality and quali-quantitative anomalies the answer given by the cell.

From the reactions that develop an immunophatology underlines could explicate under different forms that varioly are combined between them, and that we can divided as follow:

• Disprotidemia
• Pathology from immunocomplexes
• Pathology from immunoantibody
• Diffused Granulomatosi
• Amiloidosi
• Immunodepression

• Disprotidemia

Is one of most important aspect of the humoral unbalances that come true in case of Leishmaniosi, and is characterized by a total increase of the protidemia with ipoalbuminemia and ipergammaglobulinemia. The relationship Albumins/Globulins in the ill animals is inclusive between 0.10 and 0.40 ,while the normal value draws near to the unity.

• Pathology by immunocomplexes

This immunopathology has caused from the union of a soluble antigen with an precipitant antibody: the complexes that occur could fall headlong in the site of formation or they could circulate and precipitate in the various organs. In the formation of the [immunocomplexes ]soluble antigens and precipitant antibodies and only the classes immunoglobulinic IgG and IgM are able to do it. During of Leishmania the ipergammaglobulinemia is characterized by an increase of the IgG of which best part is not direct against the leishmanie.

• Pathology of autoantibodies

The selfimmunity could be definite like a immunological reactivity against the own parts that is revealed in circle with the appearance of selfantibodies. But the presence of selfantibodies does not mean that they are involved in the pathogenesis of the illness in matter.

In dogs afflicted whit Leishmaniosi smooth anti-muscle and cardiac anti-muscle antibodies have been underlined through immunofluorescence on tissue (Ceci, et all). It is probable that some substances present in the Leishmanie could induce a cruciform reactivity toward organic components. The actina and the tubulina are present in preparations of membrane of Leishmanie. Donovani, Pateraki and other have signaled the presence of antibodies antiactina and antitubulina in 263 dogs feigns from Leishmaniosi.

• Diffused granulomatosi

Many authors have signaled the presence of granulomi in several organs and tissues. Their composition is a focal accumulation of macrofagi parassitati and not surrounded by linfociti and plasmacellule in varying number, generally they are missing giant cells, necrosis central and connett ivai proliferations.

• Amiloidosi

The deposition of Amiloide is verified in the illnesses linfoproliferative, selfimmune and infectious chronic. This immunophatology is characterized amiloide substance in tissues. The Amiloide is made of proteic fibrilles that deposite in rigid structures tightly united of feltrlook. Two types of Amiloide are known: Amiloide L that is verified in illnesses linfoproliferative and Amiloide A that is verified in the infectious chronic illness and in selfimmune illness.

• Immunodepression

A depth state of immunodepression has been underlined in the course of Leishmaniosi, in particular has been underlined that, the immune answer of cellular type (Linfociti T) comes to be compromised either as quantity of active cells or as activity of the present cells in circle. Some jobs are at the moment in course to underline the reason of the block of membrane of the parasite as regards the cells linfoid.

CLINICAL DIAGNOSIS

Many clinical demonstrations of the Leishmaniosi are tied up more to the immune answer than to a precise direct action of the parasite. Often two forms of illness are quoted: they have brought again two forms of illness often: the acute form and the chronic form. The distinction between the two forms and the confinements between them are not so clean and precise and close to subacute forms, that evolve in the turn of weeks, time and chronic forms, with long lapses of months, chronic forms exist that become acute suddenly whose symptoms are tied up more to the secondary immunitary answer than to the primary infection.

A clinical acute form has been underlined in association with fever with nervous symptoms and rapid death, which on the base of the result is confused often with the nervous distemper. It could be limitative and superficial to define the canine Leishmaniosi as a chronic form since the chronic forms could be different and not all the symptoms, revealed are present contemporally.

• Fever

In the dog fever is signaled frequently and with elevated temperatures. But there could be periods of intermittent fever that can not be however appreciated.

• Skin

Four types of cutaneous macroscopic and microscopic lesions are described:

• Dry exfoliative not suppurative dermatitis

• Ulcerative dermatosis

• Nodular dermatitis

• Pimply sterile dermatitis

Common characteristic of these forms is the absence of itch. The cutaneous interest has resulted present in the 72% of the sick subjects and the more common form is the not suppurative dermatitis. This is revealed with rarefaction of the hair and seborrea dry, with symmetrical areas of alopecia: the lesions on the head are characterized by the classical "lunette" glasses in the periocular zone.Olso the limbs have to level of the bony prominences are often with a characteristic presence of furfuriformi flakes. The ulcerative form strikes the 20% around of the subjects with localizations on the legs and to level of the bony prominences. The growth of the fingernails, ornicogrifosi, in some cases is considered an inflammatory expression of the ungueal matrix provoked by the action of parasites. The immunodepression caused by Leishmaniosi could favor the coming demodicosi also in adult dogs.

• Eye

The ocular symptomatology could accompany the infection from Leishmanie or the immunologic reactions to it tied up, and sometimes is the only symptom for which the dog is led to the veterinarian. The lesions could-be primary- directly got by the parasite or secondary- tied up to the immunitary answer. In the Mastiff we have observed a few times a palpebral interest with exfoliation, thath causes the classical lunette, it is much more frequent a palpebral interest with suppurative dermatitis of the eyelids. Very frequent the cheratocongiuntiviti dry from Leishmania, in case of which a lesion could be underlined to change of the retina with separation.

• Reticoloendoteliale system

The limph nodes result thickened in almost all the subjects, while in those that they do not present cutaneous interest, but only renal involvement, the adenomegalia is absent. The [linfonodi prescapolari] is generally more increased of the poplitei. The spleenomegalia is not a constant report.

• Skeletal apparatus

The symptom limp have been many times observed in positive subjects. The pain is associated to the articular regions with edema and intraarticolare pouring.

• Blood

The anemia is a clinical frequent sign in course of canine Leishmaniosi (in the Mastiff it is present in the 80% of the positive subjects). The piastrinopenia is present but the number of the trombociti circolanti doesn't goes down to the 40000* mm3, value that explains the nasal hemorrhages (epistassi); of the observed subjects only the 20% has presented phenomenons of epistassi.

• Bowel

Often between the symptoms ofLeishmaniosi are signaled diarroic phenomenons tied up to parasite infiltrations of the foil to level of the small and big bowel.

• Kidney

The renal damages are frequent and visible either from the chemical point of view or the anatomopathological one. The proteinuria is a present clinical sign, and also if not accompanied by increases of the urea and of creatinina it is index of renal damage. Best loss of proteins is tied up to phenomenons of glomerulonefrite membrane proliferative. The proteinuria could be as massive to provoke a ipoprotinemia with abdominal edema, to the limbs and with retinic separation.

ASPECIFIC EXAMINATIONS

Emocromocitometrico examination
It is the most aspecific examination than those in course of Leishmaniosi and it is done in all the laboratory proofs. In course ofLeishmaniosi anemias normocitarie and normocromiche have been underlined (53% of cases Lubas and others). The typical tricitopenia of the visceral human forms is missing and there is a leucocitosi of secondary cutaneous lesions:' renal or of other organs. At the hematic graze there is a frequent microagglutinazione.

Eritrosedimentation speed (VES)
The eritrosedimentation speed is a very generic aspecific test which is based on the measurement of the rapidity with which the emazie sedimentano in the plasma in a taking made with anticoagulante (citrato). In course of inflammatory, infectious, tumoral processes, the speed of fall in the plasma increases.

The normal value in a subject is of2-4 mm in the first hour. In course ofLeishmaniosi it arrives to 50-100 mm.

Formogelificazione or reaction of Gate

It is a proof of colloidal weakness of the serum and therefore his positiveness depends on an altered distribution and quantity of the seric proteins. The serum of weakness animals put into contact of formalina at 40% in the 70% of the cases assumes a milky color and gels so turning 30 minutes for which turning upside-down the test-tube does not escape material.

Total proteins and elettroforesi of serum

The total proteins in course of Leishmaniosi appear greater than 8 gldl and such increase concerns the globulinic fraction exclusively. The relationship Albumins/ Gobuline lowers notably and descends under the 0.5, in course of amiloidosi the quantity of total proteins is under the norm (less then 6 gldl).

The albumin is limited either relatively to the globulinic increase and of total proteins or in absolute sense, tied up either to the reduced enteric absorption or to do decreased hepatic synthesis.

Urines of examination

The more evident characteristic in case ofLeishmaniosi in the examination of the urines is the strong proteinuria that is underlined in the best part of the cases (in around the 80% of positive subjects).

The examination of urines has a clinical importance and it has no indications on the renal damage itself.

Azotemia and creatininemia

The serologic dosing of urea and creatinina offers information on the renal conditions of the patient and must never be separated from the examination of the urines, of the determination of the total proteins, of the elettroforesi of the serum and of the proof of formalgelificazione.

SPECIFIC EXAMINATION

Serologic examinations

They are examinations based on the ability of the soluble antigen to react to the presence of antibodies present in the serum. The reaction is put into evidence through various laboratory methodics: indirect emoagglutinazione, E.L.LS.A, indirect immunofluorescenza. Some of these methodics must be performed necessarily in laboratories equipped with fluorescence microscopes, with microtester readers whose cost is not within all surgeries reach. In commerce there are some tests exploit the E.L.L S.A. methodic and of serum migration on supports of nitrocellulosa, which allow to underline the pathology. An other methodic used in the diagnosis is the P.c.R. (polimerase chain reaction) she exploits a method of amplification of the DNA. By withdrawing small quantity of organic material specific antibodies are underlined.

Biological examinations

They are easy to execute and in case of positiveness they constitute the only examination that reveals the absolute safety of the infection.

The biological examinations easier manuality are the aspiration of linfonodi through thin needle, the direct impression or for curettage from ulcerative lesions, the impression of the derma cutaneous after taking with scissors and the medullae biopsy.

All the methodics foresee the colouring of the withdrawn material crawled on slied, with t.1CG or NBM and the reading to the optic microscope to 1000 enlargements with visualization ofLeishmanie.

Cultural examinations

They foresee the growth of material withdrawn from some subjects on cultural ground maintained at a temperature between 22 and 28 degrees centigrade and numerous passages on cool grounds with optimum at 25°C.

LEISHMANIOSI THERAPY

In the last years the development of new therapeutic protocols for the treatment of the Lishmaniosi has been ofthe most interesting sectors of search of either human or vrterinary medicine. The main targets which the new medicines must aim in order to fight the Lishmaniosi are the study of the physiological,biochemistral aspects, and the parasite guest interaction with the relative immunitaria answer.

At the beginning of the third millennium an effective inexpensive medicine doesn't exist, without collateral effects and easy to administer.

ALLOPURINOLO

• Mechanism of action
Similar to ipoxantina, it is the idrolizatall of allopurinolo riboside. As such it is incorporated into R.N.A. of parasite interfering with the proteinic synthesis.

• Advantages
Sinergy of action with the antimoniato ofn-metilglucamina (Glucantim). The association is commonly used and seems to take to a better therapeutic effectiveness.

• Disadvantages
Collateral effects: diarrhea, anemia

Therapeutic protocols

* 20-30 [mg kg]/ Bid in association with the Glucantim then for 12 months as therapy of maintenance (Ferrer, 1999)

* 20 [mg kg] for a week per month as therapy of maintenance * 10 [mg kg]/ SID for 2-4 months (Cavaliero, 1999)

* 15 [mg kg]/ BID for 8 months in association with AnM 100 mg SID kg for subcutaneous way for 4 weeks (Demerolle, 1999)

ANFOTERRAMICINIA B (Amb)

• Mechanism of action 

Lipofilic macrolide, it is able to alter the ergosteroli of the membranes of the Leishmania modifyng its permeability.

• Advantages

It is possible to associate it with solutions of lipids in order to reduce its toxicity.

• Disadvantages

Administration intravenous nefrotossrca

Collateral effects fever vomits, anorexia, periflebiti

• Therapeutic protocol

* 0.5 mg/kg in solution of dextrose 5% three times a week until a maximum of9-12 mg kg (Noxon 1989)

* 0,5-0,8 mg kg within 15-45 seconds 3 times a week for a total dose of 8-15 mg kg. The therapies must be stopped if creatinina overcomes the 2.5 mg/dl (Lamothe, 1977).

ANFOTERRAMICINA B MICROCAPSULED IN LIPOSOMI

• Advantages

Preferred to the AmB not micro capsuled because it keeps plasmatic taller levels, the hepatic and splenic concentrations are elevated.

• Disadvantages Expensive

• Therapeutic protocol

* 3 mg SID kg for intravenous way for 5 days to repeat at 10° a day from the beginning of the therapy (Olive 1995)

AMMINOSIDINA

• Mechanism of action

Amino glucoside that reaches intracellular concentrations by interacting with the subunities ribosomali provoking an anomalous proteic synthesis.

• Advantages

Sinergy of action if associated with the stibiogluconato of sodium (Thakur, 1995) or with AnM (Belloli 1995)

• Disadvantages

Nephrotoxicity and ototoxicity (reversing).

• Therapeutic protocol

• 5,25-10,5 BID mg-kg subcutaneus for 3 weeks (Persechino 1994-1995).

• 10 SID mg-kg subcutaneus for 4 weeks (poles 1995).

• 3.5 BID mg-kg subcutaneus for 3 weeks in association with AnM (20 SID mg-kg) (Oliva 1998).

• 3 SID mg-kg for 4 weeks in association with Allopurinolo (Albanese, Crimaldi 2000).

ANTIMONIALI

The most used medicine for the therapy in commerce the stibiogluconato of sodium and the antimoniato of n-metilglucamina (AnM).

• Mechanism of action

They inihbit some enzymes giving place to a highly toxic form in the body of the Leishmania.

• Advantages

Rapid clinical improvement, sinergy of action with other medicines.

• Disadvantages

Duration of the prolonged treatment because brief periods or insufficient dosing could give forms of Leishmania resistant to the therapies.

• Collateral effects

Pain in the point of inoculation and local tissue reactions.

• Therapeutic protocol

• 75 SID mg-kg for subcutaneous way in association with the allopurinolo for 20 days. (Ferrer 1997).

• 50 BID mg-kg to repeat for 4 weeks up to the normalization of the clinical chart (Oliva 1998).

• 75-100 BID mg-kg for 60-90 days (pennisi 2000).

• 100mg-kg SID for 4 weeks in association with Allopurinolo 15 BID mg-kg to continue for 8 months after the suspension of AnM.

ATOVAQUONE

• Mechanism of action

Endowed with antiprotozoaria activity prohibitipg the mitocondTiale activity of the parasite.

• Advantages

Oral administration; scarce toxicity; carried with the liposomi

• Disadvantages Expensive

• Therapeutic protocol

Experimental' in the hamster 100 mg-kg for oral way for 3 weeks Experimental' in the mouse 0,04-0,32 mg-kg for intravenous way

AZOLI AND ALLILAMINE

• Mechanism of action

They interfere in the biosynthesis of parts of the parasitic cells.

• Advantages

Oral administration;sinergy of action between azoli and allilamine.

• Disadvantages

Hepatotoxicity in the dog different sensibility between the various kind ofLeishmanie.

• Therapeutic protocol

• Terbifamina: 500 mg a day for 4 weeks with parasite nagativity in the 80% of the cases

• Ketoconazolo (dog): 7 mg-kg oral way for 40/90 days

CHINOLONICI

• Mechanism of action

They inhibit DNA-girasi

• Advantages

Facility of administration; sinergy of action with the IFN-gamma (g interferone)

• Disadvantages

Anecdotal signaling

• Therapeutic protocol

Enrofloxacina: lOmg-kg SID for oral way

METRONIDAZOLO

• Mechanism of action

Arrest of the synthesis of the nucleic acids.

• Advantages

Oral administration

• Disadvantages

Anecdotal signaling (Pennisi)

• Therapeutic protocol

25 SID mg-kg in association with spiramicina 150,000 Ur. Ikg for 3 months.

MILTEFOSINA

• Mechanism of action

Similar to the fosfocolina. Experimental in the man and in the mouse

• Advantages

Oral administration

• Disadvantages

Collateral effects (gastroenteriti); expensive

• Therapeutic protocol

. 100- 150 mg to the of for oral street for 4 weeks (Sundar, 1999; Murray 2000) clinical recovery and [parassitologia] in the 97% of the cases.

PENTAMIDINA

• Mechanism of action

It damages the DNA of the Leishmania

• Advantages

Used in patients with resistance to the AnM

• Disadvantages

Hematic transitorytakes, slow elimination; istolesiva if administered for by intramuscolare

• Therapeutic protocol

. 4mg-kg for by intramuscolare at alternate days for 15 administrations in total. (Lamothe)

PHITOTERAPICI

In many African countries and Asians have been used plants whose active principle have been effective in course of Leishmania.

The amarogentina is a glucoside isolated in India and have been used in association with the liposomi giving leishmanicida action.

The garlic in association with AnM in experimental seem gave positive results in mice, besides garlic gave an immune answer. (Ghazanfari, 2000)

IMMUNOTHERAPY

• Vaccination

Experiments of vaccinations have been made in associatin with parasitic antigens with IFN in the mouse have proven the validity and provoked an effective protection in course of

experimental infection in the mouse., "

Vaccination proofs have been done by using Leishmania infantum sterilized at 100% antigens, in association with AnM which have been used as vectors in dogs therapy.

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