PhD

The nature of embryomortality in hatcheries and the possible causes for
embryopathies were studied. Experimental studies were conducted by
inoculating oxytetracycline, Ochratoxin A, Citrinin and a combination of
Ochratoxin A and Citrinin and avian influensa virus. Ultra-structural studies
were conducted to elucidate the subcellular changes in the liver and kidneys
of the embryo after administration of the mycotoxins.
The various abnormalities noticed were early embryonic death, curled
embryos, dead in shell, live sticky embryos, oedema of head and neck,
omphalitis and septicaemia, gastroschisis with herniation of yolk sac,
coelosoma,
dwarfs,
curled
toe,
microphthalmia,
anophthalmia,
brachycephaly, parrot beak, crossed beak, brachygnathia, streptosomia and a
few cases of diprosopus, polymelia, ectromelia, thoracomelia, phocomelia and
duplicatus. Sixty per cent of these embryos had double or multiple defects.
A total of 115 bacterial isolates was made from a total of 150 embryos
and 26 samples showed the presence of more than one type of organism. The
organism
isolated
wer
staphylococcus,
Corynebacterium,
Bacillus,
Micrococcus, Aerococcus, Proteus, Pseudomonas, Alkaligenus, Aeromonas
and Coliforms. Contamination of the hatching eggs could be the cause for
these infections.
When oxytetracycline was givan to 8 day old embryos only 47.5%
survived up to 21 days. The mean average weight of the embryos was
significantly lower than that of controls. Degenerative changes were noticed in
liver, kidney and digestive organs. Generalized oedema was observed. There
was interference in chondrogenesis, osteogenesis and calcification.
In the group of embryos inoculated with ochratoxin A 75% died. These
embryos showed imperfect organogenesis. There was marked degeneration
and necrosis of the cells of various developing organs. The ultra-structural
changes in the liver and kidneys were mainly characterized by damage to the
membrane systems. The mitochondria showed changes varying from swelling
to severe destruction of the organellar structures. There was fragmentation
and destruction of the rough endoplasmic reticulam. Necleus was severaly
damaged. Moderate destruction of the proximal convoluted tubules was
noticed. In the glomeruli, there was an increase in the fenestrated nature of
the endothelial surface of the basement membrane and damage to the foot-
processes of the podocytes.
There was 67.5% mortality in the citrinin treated group. Significant
reduction in the weight of the embryos was noticed. There was degeneration
and necrosis in the kidney, liver and other organs. Calcified foci were
observed in the kidney and liver. The ultra-structural changes in the liver
were swelling of mitochondria with damaged cristae and ring shaped nucleoli
with desegregation of nucleolonema and granular components. In the kidney
there was severe damage to the epithelial cells of the tubules. Chromatolysis
and lysis of nuclear membrane were seen. Slight swelling with enlargement of
the outer compartment to complete lysis of the cristae was noticed in the
mitochondria. In glomeruli there was swelling of mitochondria of the
podocytes, mesangial cells, endothelial cells and cells lining the Bowman’s
capsule.
In the group which were treated with citrinin and ochratoxin A there
was 87.5% mortality and there was reduction in weight of the embyos. The
survived embryos showed malformation like cranioschisis, herniation of yolk,
curled toe and eventration of viscera. Histologically there was severe
degeneration and necrosis of all organs and there was interference in the
organogenesis.
The ultra-structural changes were more severe than those observed in
ochratoxin A and citrinin toxicity individually. Ring shaped and giant
mitochondria were observed. There was degranulation of ribosomes. In the
Kupffer cells there was increased number of lysosomes. The organellar
damage was more severe in the proximal and distal convoluted tubules. The
basement membrane of the glomeruli was found to have lost its homogenous
nature and there was fusion of foot-processes of the podocytes.
When the embryos were inoculated with avian influenza virus there was
general vascular, cellular and degenerative changes. Heterophils were not
present eventhough there was general congestion.
The cytotoxic action of the virus was evident by the degenerative and
necrotic changes of the parenchymatous organs. There was fragmentation
and lysis of myocardium. Neuronal degeneration and liquifactive necrosis
were seen in the brain.
FINDINGS :
This study was conducted to assess the nature of embryo-mortality in
hatcheries, to find out the possible causes and to investigate the
pathoanatomical features of embryopathies. A total of 5440 embryos (out of
32,700 eggs set) which failed to hatch on the 21st day was studied
pathomorphologically. Out of these, 150 embryos which showed signs of
infection were subjected to bacteriological investigation. In order to ascertain
the role of various agents in causing mortality and malformations in the
embryo, experimental studies were conducted by inoculating Oxytetracycline,
Ochratoxin A, Citrinin, a combination of Ochratoxin A and Citrinin and
avian influenza virus. Ultra-structural studies were conducted to elucidate the
sub-cellular changes in the liver and kidneys of the embryo after
administration of Ochratoxin A, Citrinin and a combination of Ochratoxin A
and Citrinin.
The various abnormalities noticed were early embryonic death, curled
embryos, dead in shell, live sticky embryos, oedema of head and neck,
omphalitis and septicaemia, gastroschisis with herniation of yolk sac,
coelosoma,
dwarfs,
curled
toe,
microphthalmia,
anophthalmia,
brachycephaly, parrot beak, crossed beak, brachygnathia and streptosomia.
A few cased of diprosopus, polymelia, ectromelia, thoracomelia, phocomelia
and duplicatus were also encountered.
Sixty per cent of the embryos examined had double or multiple defects.
Twenty-seven per cent had oedema in the region of the head and neck. In
these, histologically there was fragmentation and mylosis of fibres. Such
changes could be due to postural abnormality, toxicosis or vitamin E
deficiency. Thirty-eight per cent of cases because of their poor development
indicated early embryonic death, that is before 10 days of incubation. Ten per
cent of the embryos were small in size and had a curled appearance. The size
of the embryo indicated their death had occurred between 10th and 18th days
of incubation. Dwarf embryos constituted 11.3% (Total 615). Out of these 55
showed extreme shortness of legs and 70 cases revealed haemorrhages in the
liver, kidney and heart. Histologically there was degeneration and necrosis of
these organs. Among these dwarf embryos 22 numbers had generalized
oedema.
Five per cent of the cases revealed omphalitis and lesions of general
septicaemia. This was seen in embryos where there was herniation of yolk sac
as well in those embryos where the yolk was retained within the body.
Hundred and fifty of these embryos were subjected to bacteriological
examination. A total of 115 bacterial isolates was made and 26 samples
showed the presence of more than one type of organism. The organisms
isolated were Staphylococcus, Corvnebacterium, Bacillus, Micrococcus,
Aerococcus, Coliforms, Proteus, Pseudomonas, Alkaligenus and Aeromonas.
Contamination of the hatching eggs could be the factor for yolk sac infections
and septicaemia.
Fifteen per cent of the embryos were classified as ‘dead in shell’. They
did not reveal any malformations except for moderate degree of congestion
and degeneration changes in the liver and kidney. Embryos which were
classified as ‘live sticky’ constituted 20%. Coelosoma (7.87%) was
characterized by envtration of viscera which varied from loops of intestines to
entire viscera seen outside the body.
The abnormalities and deformities of bone and cartilage encountered
were beak abnormalities like parrot beak (2.3%), crossed beak (3.69%),
brachygnathia (0.28%), agnathia (0.2%), micromelia (2.5%), polymelia
(0.02%), ectromelia (0.02%), curled toe (4.49%), brachycephaly (2.5%) and
cranioschisis with encephalocoele (11.03%). In cranioschisis, there was partial
failure of the fusion of cranium and herniation of the brain. In such cases
brain was malacic with neuronal degeneration. It was suggested that many of
these developmental defects could be due to nutritional factors or due to
toxicants. In 2430 embryos (45%) various types of malpositions were
observed. They were single or multiple.
The other malformations encountered were few in number. When oxy-
tetracycline was given to 8 day old embryos, only 47.5% survived at 21 days.
The rest died during various stages of incubation. The mean average weight
of the embryos that survived was significantly lower that of controls.
Degenerative changes were noticed in liver, kidney and digestive organs.
Generalized oedema was observed. There was interference in chondrogenesis
and osteogenesis and calcification. It was postulated that these might have
contributed to the dystrophic changes in the bones.
In the group of embryos inoculated with Ochratoxin A, 75% died and
showed a marked reduction in size and weight. The embryos which were dead
at various stages of incubation showed imperfect organogenesis. There was
marked degeneration and necrosis of the cells of various developing organs.
Aberration of development was seen in the growing bones resulting even in
cranioschisis seen in the eye. In the retina, there was detachment or folding of
the ganglionic layer. Degeneration of the lymphoid elements of the developing
thymus and bursa of Fabricius was also noticed. The ultra-structural changes
in the liver and kidneys were mainly characterized by damage to the
membrane system. The mitochondria showed changed varying from swelling
to severe destruction of the organellar structures. There was fragmentation
and de-granulation of the rough endoplasmic reticulum. Lysosomes were
present. Nuclear changes reflected the degree of injury to the cells. Nuclear
changes reflected the degree of injury to the cells. There was moderate
destruction of the brush border of the proximal convoluted tubule. The
changes in the glomeruli were mainly confined to the basement membrane
and podocytes. There was an increase in the fenestrated nature of the
endothelial surface of the basement membrane and damage to the foot
process of the podocytes. It was suggested that since ochratoxin A was a
known inhibitor of mitochondrial transport systems many of the structural
alterations could be correlated to this metabolic change and the resultant
disturbed differentiation could result in imperfect organogenesis including
osteodystrophic changes.
There was 67.5% mortality when the embryos were inoculated with
citrinin. The embryos that survived had significantly lower weight than
controls. The embryos which died before 21 days showed general congestion,
degeneration and necrosis of various organs. The developing bones were
apparently normal. In the brain there was slight oedema of the dienephalon.
The embryos that survived up to 21 days also showed degeneration and
necrotic changes in the kidney and liver. Calcified foci were also noticed in
these organs. In three cases the epithelial cells of the ciliary body of the eye
showed slight degeneration between the conjunctivo-vascular layer of the
ciliary bodies and sclerotic coat. Slight oedema and degeneration of the brain
was also noticed.
The ultra-structural changes in the liver associated with citrinin toxicity
were mitochondrial swelling with damaged cristae, ring shaped nucleoli with
desegregation of nucleolenoma and granular components, partial lysis of
plasma membrane and presence of debri in the bile canaliculi. In the kidney
the epithelial cells of the tubules showed the severest form of damage with
some cells showing organellar changes indicative of advanced necrobiotic
changes. Chromatolysis was partial or complete with occasional lysis of
nuclear membrane. Slight swelling with enlargement of the outer
compartment to complete lysis of the cristae was noticed in the mitochondria.
In the glomeruli there was mild to severe swelling of mitochondria of
podocytes, mesangial cells, endothelial cells and cells lining the Bowman’s
capsule. There was occasional fusion of podocyte foot-processes.
There was 87.5% mortality of embryos in groups inoculated with the
combination of Ochratoxin A and citrinin. There was reduction in weight of
embryos that survived up to 21 days and the malformations noticed were
cranioschisis, herniation of yolk sac, curled toe and eventration of viscera.
General oedema was also observed. The histological changes in the liver,
kidney, alimentary organs, thymus and bursa of Fabricius were severe
degeneration and necrosis. Interference in the chondrogenesis, osteogenesis,
ossification and calcification was noticed in the bones. The ill-developed
cranial bones resulted in imperfect fusion resulting in herniation of brain.
There was oedema of brain and degeneration of neurons. In the eye there was
degeneration of the ganglionic cell layers of the retina and epithelial cells of
the ciliary body. The ultra-structural changes seen in the liver and kidney
were more severe than what were observed in ochratoxin A and citrinin
toxicity individually. Ring shaped and giant mitochondria were observed.
There was de-granulation of ribosomes. In the Kupffer cells there was
increased number of lysosomes. The organellar damage was more severe in
the proximal and distal convoluted tubules than in other parts of the kidney.
The basement membrane of the glomeruli was found to have lost its
homogenous nature and there was fusion of foot processes of the podocytes. It
was observed that the toxicity of Ochratoxin A and citrinin was
synergistically augmented by their simultaneous administration.
The response of the chick embryo to the inoculation of avian influenza
virus was characterized by general vascular, cellular and degenerative
changes. Eventhough there was general congestion, heterophilic infiltration
was not a characteristic feature. It could not be ascertained from this study
whether the failure of heterophilic emigration was due to the functional
incompetence of these cells because of the early stage of the embryo or due to
the inability of the embryo to respond to the chemical nediators or to produce
the chemical mediators themselves. The cytotoxic action of the virus was
evident by the degenerative and necrotic changes of the parenchymatous
organs. The myocardium was fragmented and showed myolysis. There was
general oedema. Neuronal degeneration and liquifactive necrosis were seen in
the brain.
This study was helped to elucidate the pathomorphology and some
aspects and the etiology of embryomortality in chicken. The histological and
ultra-structural aspects have helped to identify the cellular and sub-cellular
events associated with mortality and teratological defects in the chicken
embryo.