Professional Documents
Culture Documents
Introduction:
Avian mycoplasmosis is a composite term referring to a group of infectious and
contagious diseases afecting chickens, turkeys, ducks, geese and partridge caused
by diferent species of Mycoplasma.
Diferent Avian Mycoplasma infections:
Disease
Avian
Mycoplasmas
Species
afected
Importance
Chronic respiratory
disease M. gallisepticum
Chicken and
turkeys
++++
Infectious sinusitis Turkeys ++++
Infectious synovitis M. synoviae
Chicken and
turkeys
+++
M. meleagridis infection M. meleagridis Turkeys +++
M. ioae infection M. iowae Turkeys +
M. immitans infection M. immitans !ucks and geese "
Historical Informations:
1-Mycoplasmas ere #rst identi#ed in $%&% as the etiologic agent of the contagious
bovine pleuropneumonia 'C())* and thereafter, all similar agents ere named
pleuropneumonia+like ')),-+like* organisms.
2-Avian mycoplasma as #rst described in turkeys in $&./ 'infectious sinusitis of
turkeys* and in chickens in $&0/ 'chronic respiratory disease + C1!*. The cause of
them as later named as M.gallisepticum 'M2*.
3-Infectious synovitis caused by M.synoviae 'M3* as #rst reported ith arthritic
involvement from the decades of 45 6 /5 in broiler 7ocks, but it as only in the 859s
that the respiratory disease caused by M3 as described.
4-The #rst evidence of airsaculitis in day+old poults by mycoplasmas other than M2
caused by M.meleagridis 'MM*, a mycoplasma infecting turkeys and other birds, but
not chickens as discovered for the $
st
time in the decades of %5.
-M. ioae 'MI*, a mycoplasma of natural occurrence in turkeys as reported in
chickens and other birds. It as discovered for the $
st
time in the decades of &5.
!-In the end of the decade &5, M. immitans as discovered and found that it is
serologically cross+reacts ith M2, but is not yet isolated from poultry.
3ince $&4:, avian mycoplasmosis is considered a signi#cant problem in chicken
7ocks in ;apan and in other Asian countries. In ;apan, M.gallisepticum 'M2* and M.
synoviae 'M3* infections ere con#rmed etiologically in chicken 7ocks afected ith
respiratory disease or synovitis in $&/. and $&80, respectively. In other Asian
countries, including Indonesia, the )eople<s 1epublic of China, =orea, Malaysia,
)hilippines, Taipei China and Thailand, the occurrence of mycoplasmosis in chicken
7ocks is recogni>ed serologically or etiologically. Adverse atmospheric and
environmental conditions, in addition to mi?ed infections of bacterial or viral origin,
play an important role in the spread of M2 and M3 ithin chicken 7ocks or in the
induction of clinical respiratory mycoplasmosis. 3erological tests are important in
determining and monitoring the mycoplasmal infection status of chicken 7ocks. The
establishment of mycoplasma+free breeding stocks is recogni>ed as essential for the
control of avian mycoplasmosis. To eliminate the transmission of M2 to the egg,
treatment of infected breeder 7ocks or their progeny ith anti+mycoplasmal
antibiotics as efective in considerably reducing the infection rate but not in entirely
eliminating M2 infection. The pre+incubation heat treatment of chicken hatching eggs
has proved an efective procedure for establishing M2+ and M3+free breeding stocks
in ;apan. @accination against M2 infection has been practiced successfully in ;apan
and other countries.
"conomic Importance:
1-!ecreased egg production 'Mycoplasma+infected chicken lays $4.8 eggs less than
healthy one*. This is due to poor o?ygen supply reAuired for metabolism.
2-1educed egg Auality. This is due to poor nutrient utili>ation due to poor
metabolism.
3-)oor hatchability 'Mycoplasma+infected breeders give 8+$5B loer hatchability
than healthy one*. This is due to high rate of embryonic mortality and culling of day+
old birds.
4-)oor feed eCciency 'Mycoplasma+infected broiler give 8B less feed conversion 6
$5B :+eight gain less than a healthy one*.
-Increased mortality 'Mycoplasma+infected broiler give $4B higher mortality than
healthy one*. This is due to the predisposing efect of Mycoplasma to other
pathogens.
!-Increased carcass condemnations in meat+type birds due to severe emaciation.
#-Transient immunosuppression particularly for M3 infection. M3 as proven to afect
the humoral response of chicks vaccinated ith a ,a 3ota strain of D!@.
$-Increased medication costs.
"tiolo%y &'eneral(:
Diferent species of Mycoplasma:
Avian
Mycoplasmas
Disease Species afected
Importanc
e
M. gallisepticum
Chronic respiratory
disease
Chicken and
turkeys
++++
Infectious sinusitis Turkeys ++++
M. synoviae Infectious synovitis
Chicken and
turkeys
+++
M. meleagridis M. meleagridis infection Turkeys +++
M. iowae M. ioae infection Turkeys +
M. immitans M. immitans infection !ucks and geese "
Mycoplasmas are microorganisms belonging to 1ickettsia group and having the
folloing characteristic featuresE
1-)*ey resem+les +acteria in:
+Faving to types of nucleic acids '!DA and 1DA*.
+Faving triple layered plasma or cell membrane.
+3ensitivity to antibiotics and chemotherapeutics.
+Their capability for cultivation on arti#cial media ')),- mediaG and Modi#ed
Chanock<s medium* producing round colonies ith central nipple 'fried egg
appearanceGG* that seen via the inverted microscope. The agar container should
be tightly closed and incubated at 08 C for .+0 eeks in a high humid atmosphere
ith $5B C-
.
tension. The incubation apparatus may be Candle ;ar or C-
.
incubator.
G)),- medium is a speci#c medium for Mycoplasma spp. containing bile salts,
thallium acetate and avian serum '$5+.5B*.
GG)athogenic avian mycoplasmas strains gro on solid media in to types of colonies.
The small ones are typical )),-H the large ones seem to occupy an intermediate
position beteen )),- and bacteria. The elements constituting the large colonies
must be the so+called ,elson +odies.
-t*er Mycoplasma Media
Modi.ed "d/ard0s Media &1a*ane(
(aseE
(acto Feart Infusion (roth $..B
(acto Ieast J?tract 5.4B
(acto )eptone 5.4B
DaCl 5.5.4B
Da+!DA 5.55.B
pF is adKusted to % and autoclaved. May be stored at room temperature for : eeks.
A!! 'to #nal conc.*
=.F)-: 5.$8B
2lucose $.5B
Thallium Acetate 5.5.4B
)enicillin 455uLml
Forse 3erum $5B
Da+!DA 5.55.4B
pF to 8.%
:B culture is added and gron statically at 08MC for $%+.5 hours to A./5
A# a%ar &21(
2ibco base 'A8* :&.4 g
!N $.08.4 ml
pF to 4.4
Autoclave
Forse 3erum 055 ml
Ieast J?tract $4 ml
C@A 8.4 ml
Orea '$5B* $4 ml
Cysteine ':B* 0.84 ml
amp., fung., nyst. 5.4 ml
)enicillin 2 $ ml
123
123 3ase
)),- ...$ g
!N $585 ml
)henol 1ed $.4 ml
pF to 4.4
!ispense into $55 ml bottles ith P 85ml L bottle and autoclave
AddLbottle
Forse 3erum .5 ml
Ieast J?tract $5 ml
C@A 5.4 ml
Cysteine 5..4 ml
Orea 5.: ml
amp., fung., nyst. 5.54 ml
)enicillin 2 5.$ ml
2-)*ey resem+les viruses inE Their capability for cultivation in embryonated
chicken eggs mostly via yolk sac inoculation at the 4
th
day post+incubation and rarely
via CAM at the $5
th
day post+incubation.
3-Have not cell /all: This makes them resistant to antimicrobials acting on the cell
all as penicillins ')enicillin 2 sod, )enicillin 2 pot, aminopenicillins including
ampicillin 6 amo?ycillin* and cephalosporins 'Cefradin, Cefatre?yl, cefota?ime sod.,
cefopyra>one sod. and Ceftifur sod.*.
4-Intracellular and e4tracellular situation: 3ome strains are e?tracellular
agents, but some others are obligatory intracellular parasites, hereas all other
mycoplasmas are facultative intracellular organisms.
-5ra%ile microor%anism: cannot still alive outside the living body more than very
fe days so, carrier birds are essential to the epidemiology of MycoplasmasQ
diseases.
6*ronic 7espiratory Disease
Mycoplasma gallisepticum infection8
9:;<=> ?@ ABCD=> ?EFGH=> IJD=>
I-Synonyms: M2 R Mycoplasma gallisepticum infection R Chronic respiratory
disease R C1! 'Chicken* R Air sac cold R Air+sacculitis.
II-De.nition: An infectious 6 contagious disease of chickensQ, turkeys and rarely
other poultry species characteri>ed by respiratory rales, coughing and nasal
discharge. The clinical signs are slo to develop and the disease has a long course.
The disease is present in Jgypt.
III-"tiolo%y: In addition to all concepts under the "tiolo%y &'eneral(E
$+M.gallisepticum is the cause of uncomplicated airsaculitis. 3mall coccoid #lterable
organism about 5.4 O in diameter.
.+M.gallisepticum my produce disease by itself or its action may be complicated by
the folloing factors.
+E.coli and Hemophilius paragallinarum in chickens.
+@irus infection or vaccination of D! and I( in chickens.
+3tress factors 6 environmental disorders as high dust 6 ammonia levels in
chickens and turkeys.
+Bordetella avium infection and T1T infection in turkeys.
IK-"piLootiolo%y:
1-1ey points of epiLootiolo%y of M.gallisepticum :
$+Carrier birds are essential source of infection and represent the most important key
point to the epi>ootiology of M.gallisepticum infections.
.+@ertical transmission of M.gallisepticum.
0+Intesi#cation of poultry farms.
:+@iability of M.gallisepticum in organic mattersE
M.gallisepticum remained viable in chicken feces for $+0 days at .5MC, on muslin
cloth for 0 days at .5MC or $ day at 08MC, and in egg yolk for $% ks at 08MC or /
ks at .5MC. It as found that M2 could survive in the human nasal passage for .:
hrsH on stra, cotton, and rubber for . daysH on human hair for 0 daysH and on
feathers for .+: days.
2-Mode of Infection and )ransmission:
1-Kertical transmission &in-ovoM transovarian(: The highest rates occur during
the acute phase of the disease hen M2 levels in the respiratory tract peaked. )eak
egg transmission occurs : + :.4 ks post+infection in about .4+45B of the laid eggs.
2-HoriLontal transmission:
A-!irect contact of the susceptible birds ith infected clinical or sub+clinical birds
resulting in high infection andLor disease prevalence ithin 7ocks.
3-Indirect contact ith the fomites as contaminated airborne dust, droplets or
feathers, coupled ith sub+optimal biosecurity and personnel practices.
C-The upper respiratory tract andLor conKunctiva are the route of infections in
aerosols or droplets.
Fori>ontal spread of M.gallisepticum ithin a chicken 7ock is described in four
phasesE
i-N*ase-1 &latent p*ase( '$.+.$ days* before antibody as #rst detected in
inoculated birds.
ii-N*ase-2, a period '$+.$ days* in hich infection gradually appeared in 4+$5B of
the population.
iii-N*ase-3, a period '8+0. days* in hich &5+&4B of the remaining population
developed antibody.
iv-N*ase-4 &terminal p*ase( '0+$& days* in hich the remainder of the
population became positive.
3-Suscepti+ility: Chickens, turkeys and rarely other poultry species. (irds of :+
ks or older are the most susceptible to the disease.
4--ccurrence: The disease is idespread in all countries. It represents an
impact problem in poultry industry from the national point of vie in Jgypt.
-Seasonal IncidenceE It is a seasonal disease, more often seen in inter and
fall.
!-Incu+ation Neriod: The I) of M.gallisepticum infection varies from /+.$ days
hile in infectious sinusitis, it varies from /+$5 days.
K-6linical Symptoms:
1-6ommercial +roiler c*icOens: The disease is chronic in nature and
characteri>ed byE
a+Dasal odorless discharge ith foamy ocular e?udates and sollen periorbital
sinuses.
b+)ersistent coughing, snicks, tracheal rale, snee>ing and gasping.
c+,oss of appetite ith decreased feed consumptionH retarded groth and the 7ocks
become uneven in si>es ith don grade of carcasses.
d+Mortality due to uncomplicated C1! is lo. -n the other hand, in the complicated,
mortality is variable and depends on the environmental climatic conditions and the
type of secondary infection.
2-In PayersQ3reeder c*icOens: The disease is chronic in nature and characteri>ed
byE
a+Dasal and ocular discharge 'atery eyes* tracheal rale in the ind pipes, coughing,
gasping 'dyspnea*, snee>ing and shaking of the head.
b+Seed consumption drops of leading to decreased egg production '.5+05B* as ell
as loss of body eight.
c+Male birds freAuently have the most prominent signs.
d+1educed hatchability and chick viability.
e+-ccasional encephalopathy and abnormal feathering.
KI-Nostmortem lesions:
1-6*icOen:
a. Catarrhal e?udates in the upper respiratory tract.
b. The air sacs freAuently contain caseous e?udates and may present a TbeadedT
appearance.
c. (irds that have a secondary infection sho #brinous e?udates in the pericardial
sac and liver surface, in addition to the above lesions.
Sta%es of 6*ronic 7espiratory disease:
"arly sta%e of 67D
Advanced sta%e of
67D
667D
J?cess mucous in trachea. )lugs or pus in trachea.
)erihepatitis 6 pericarditis
'esp. ith secondary E.coli
infection).
Consolidated and solid
spots on lungs.
Muscles pale and 7abby
Sormation of creamy
discolored '#brinous* layer
over heat and liver. Thickness
of layer indicates the
advancement of CC1! stage.
Cheesy material in lungs
Catarrhal in7ammation
of nasal passages,
sinuses, trachea and
bronchi.
Air sacculitis i.e.
in7ammation of air sacs
-ccasionally arthritis
and salphingitis in
chickens.
(eaded or ,ymph+follicular
appearance.
Caseous e?udates in air
sacs TSoamy or 3oapyT air
sacs.
KII-Dia%nosis:
RHistory taOin%
+Adverse environmental conditions 'overcroding, poor ventilation and high
ammonia level* increase the incidence of the disease.
+Mi?ed infections especially ith E.coli are common in the #eld.
+@accination ith live virus vaccine may induce the disease.
+Chronic respiratory symptoms ith lo mortality.
+Transmission rate is relative slo i.e. very chronic disease.
R6linical si%ns S NM lesionsTTT as discussed but give a great attention to
investigate air sacs, hich are involved more e?tensive than any other disease.
RPa+oratory Dia%nosis:
a-Serolo%y:
1-Hema%%lutination
Nurpose E Femagglutinate chicken 1(Cs.
Met*od:
+Allantoic cavity 7uid 'possibly containing M.gallisepticum* from an
embryonating chicken egg that had been inoculated ith suspect material is
removed.
+5.$ ml of the 7uid is placed on glass plate and 5.$ ml of 4.5B suspension of
chicken 1(C<s is added.
+If a hemagglutinating agent is present, the 1(Cs ill clump.
GD(+ This is not a serological test. Nhile it is used as the basis for the
hemagglutination inhibition test. It detects antigen not antibody.
2-Hema%%lutination-In*i+ition test &HI(
Nurpose: To Auantitate serum antibody to a speci#c avian 'antigen*.
$. Avian pathogens hich can hemagglutinate avian 1(Cs 'hemagglutination*
include D!@, In7uen>a virus, D! virus and Adenovirus $.8H in addition,
Mycoplasmas are also capable of hemagglutinating avian 1(Cs.
.. Antibodies directed against these organisms ill inhibit hemagglutination.
Nrocedure:
a+A constant amount of hemagglutinating 'FA* antigen is added to each ell in
a microtiter plate.
b+The test serum is then placed in the #rst ell and serially diluted.
c+The plates are incubated for one hour and then chicken 1(Cs are added to
each ell. If antibody is present in the test serum the 1(Cs ill not agglutinate
ith the FA antigen.
7esults and interpretation:
a. FI Degative ells ill have a difuse sheet of agglutinated 1(Cs covering the
bottom.
b. FI )ositive ells ill have a ell+circumscribed button of un+agglutinated
1(Cs.
c+Considering that FI titers eAual or higher than $E%5 are regarded as positive
and titers beteen $E.5+$E:5 are suspicious, hereas negative titers are belo
$E.5.
3-Serum plate a%%lutination test &SNA)( U 7apid serum
a%%lutination test:
Nurpose: To Auantitate serum antibody to a speci#c avian 'antigen*.
$+Collected not immediately tested sera are stored at :MC and not fro>en.
.+The test should be carried out at room temperature ithin 8. hrs of sera
collection.
0+)rior centrifugation ill reduce nonspeci#c reactions.
Nrocedure:
a--ne volume 'appro?imately 5.5. ml* of serum is dropped on to a hite tile
folloed by one volume of stained M2 antigen.
b+The tile is rotated to ensure mi?ing.
7esults and interpretation:
$-Agglutination is indicated by clumping ithin . minutes. =non positive and
negative controls should be incorporated in the test. The antigens are available
commercially but may vary in speci#city and sensitivity from diferent
manufacturers and from batch to batch.
.-Any sera that agglutinate should be heated at 4/MC for 05 minutes and
retested. If they still react strongly, they are considered to be positive,
especially if they do so on dilution '$L: or more*.
0+A high proportion of positive sera in a 7ock '$5B or more* indicates M2
infection, especially if con#rmed by FI test or J,I3A.
:+Sor further con#rmation, the 7ock should be retested ithin a month.
4+Agglutination can be performed ith egg yolk samples and the results are
considered positive, suspicious and negative for titers eAual or higher than
$E$5, $E4 and loer than $E4, respectively.
+-Aller%ic test: An allergic intra+dermal test may be used for the diagnosis of
avian mycoplasmosis. This is done by inoculation of protein antigen intra+dermally
produces edematous selling ithin a fe or $.+.: hrs in infected bird. This test
as compared and found better than, agar gel precipitation test.
c-'ro/t* in*i+ition test: by observing inhibition of groth by discs
impregnated ith speci#c antisera against various Mycoplasma species.
d-Isolation and identi.cation of M8%allisepticum8
Specimen: e?udates of nasal cavity, trachea, air sac, Koint cavity and lung tissue
emulsion.
Medium: Modi#ed Chanock<s medium or )),- medium.
6ulture: 5.. ml. of emulsion is inoculated into liAuid medium and incubated at
08MC for $ eek. After color changes, $ drop of cultured broth is spread onto the
above agar medium and incubated at 08MC for 0+4 days under 4B C-
.
condition.
-6olony morp*olo%y: typical nipple shaped colonies 'a tiny circular mass ith a
dense raised central area USried egg appearanceV*.
Isolated colonies of M. gallisepticum is con#rmed byE
+Its capacity to agglutinate the chicken or turkey erythrocytes and biochemically
hence, it can ferment maltose and glucose but not dulcitol, lactose or salicin.
+)olymerase chain reaction ')C1* can be used to detect the seAuence of
Mycoplsma !DA.
Diferential Dia%nosis: !iferential diagnosis includes D!, I(, I,T and AI.
Nrevention and control
Nrevention: If M. gallisepticum are killed in the hatching eggs then the 7ock can
be maintained free from the disease. This is because, at least, one out of 45 eggs
laid by an infected hen contains these organisms and after hatching the infected
hatched chicks spread infection to others.
1-Hy%iene and mana%ement: Nhen the
disease has occurred management and feeding should be improved. 1educe
croding and dust in the poultry house. !ust helps in entry of E.coli as ell as
Mycoplasma spp. Nater should be good and holesome. 2reat attention should be
given to a7ato?icosis andLor ochrato?to?icosis, hich cause immunosuppression and
precipitate the disease.
2-Kaccines
-Pive vaccines:
+,ive vaccines are generally produced from the S+strain of M2, and more recently,
strains ts+$$ and /L%4, hich are apathogenic strains ith improved safety
characteristics.
+Administration of the S+strain by the intranasal or eye drop route is preferred, but
aerosol or drinking ater administration may be used.
+The eye drop method is recommended for ts+$$, hile #ne spray is recommended
for /L%4.
+)ullets are vaccinated beteen $.+$/ ks of age. A single dose is suCcient and
vaccinated birds remain permanent carriers.
+,ong+term use of the S+strain on a multi+age site results in displacement of the #eld
strain ith the vaccine strain. S+strain displaces virulent ild+type M2 strains more
eCciently than /L%4 or ts+$$, but ts+$$ is successfully used to eradicate S+strain M.
gallisepticum in multi+age commercial layers. Multi+age production sites are knon to
test serologically negative for M. gallisepticum after long+term use of /L%4.
,3-The S strain is fully virulent for turkeys.
Inactivated vaccines:
Inactivated vaccines or bacterins consist of a concentrated suspension of M.
gallisepticum organisms in an oil emulsion. These are ordinarily administered to
groing pullets at $.+$/ ks of age. They are administered subcutaneously in the
neck. Although . doses are desirable, a single dose is usually given because of cost
and labor considerations. (acterins are efective in preventing egg+production losses
6 respiratory disease, but they do not prevent infection ith ild+type M2.
3-1illin% M. gallisepticum in *atc*in%
e%%s:
'i* Feating eggs for .+0 hrs by keeping them in egg incubator, then eggs are dipped
in a atery solution of 5.5: to 5.$B tylosin tarterate or erythromycin or gentamycin,
the temperature of hich should be .+4 C 'refrigerator temperature outside free>ing
chamber*. Jggs are dipped in the solution for $5+05 min. This method causes
e?pansion of egg contents by heating to 08+0% C 'incubator temperature* and
dipping in cold antibiotic solution causes shrinkage and entry of the solution,
suCcient to kill the organisms.
'ii* M.gallisepticum in eggs can be killed by heat treatment, hich kills the organisms
but not the chick embryo. Jggs are kept in incubator ith temperature of :/ C for a
period of $.+$: hrs, then, these eggs are incubated for normal hatching in incubator.
This method has a disadvantage of loering the hatchability by %+$.B.
4-1illin% M8 %allisepticum in *atc*ed c*icOs: Tylosin is given to nely hatched
chicks by mi?ing in their drinking ater in dose of 45 mgLkg b. for : days from the
$st day of hatching.
Nro%ram of usin% )ylosin 5or Nrevention of 67D in
commercial +roilers
"fect Durat
ion
Daily amount of
tylosin for1222 +irds
A%e
1educe level of
Mycoplasmas of parental
origin 6 control hori>ontal
contamination
: days $4+.5 gm in .4 liters of
drinking ater.
1st /eeO of a%e
)rovide better resistance to
stress 6 improve feed
conversion
.+0
days
:5+/5 gm in 84 liters of
drinking ater.
4
t*
/eeO of a%eR
Nro%ram of usin% )ylosin 5or Nrevention of 67D in
commercial layer and +reeders
"fect Durati
on
Daily amount of tylosin
for1222 +irds
A%e
Felps maintain 7ock
free of Mycoplasmas,
ensure regular groth
6 increase resistance
to stress.
0+4
days
$4+.5 gm in .4 liters of
drinking ater.
1
st
/eeO of a%e
$+0
days
:5+/5 gm in 84 liters of
drinking ater.
t*
V !
t*
/eeO of
a%eR
To control level of
Mycoplasmas 6
ensure optimal
production of top
Auality chicks.
.+0
days
$55+$45 gm in .55 liters of
drinking ater.
1$
t*
V 1W
t*
/eeO of
a%eR
.+0
days
$55+$45 gm in .55 liters of
drinking ater.
"%% production
&every 2 mont*(
G -r Immediality before or after vaccination
)reatment: Nhen the disease is observed it indicates poor feeding,
management or other forms of stress.
$+Tylosin 'Atco+Tylan
, Tylan
and Tyloveto