Changes in the Differential Leukocyte Count of Chicks Inoculated with Salmonella

Studies were conducted to determine the effects of Salmonella anatum and S. heidelberg infections on the differential leukocyte counts of baby chickens. Newly hatched broiler-type chicks were inoculated in the yolk sac with suspensions of either S. anatum or S. heidelberg. At 0, 24, 48, and 72 hr after inoculation, blood was taken by heart puncture from chicks of both inoculated groups and from a group of uninoculated chicks. The averages of the leukocyte counts of three or four chicks from each group were used as the blood values for specific time intervals. The six classes of leukocytes counted were lymphocytes, monocytes, juveniles, heterophils, eosinophils, and basophils. The leukocytes classified as juveniles were immature or degenerate heterophils and were found almost exclusively in the infected chicks. Changes in heterophil, juvenile, and lymphocyte counts were affected by both the number of cells in the inoculum (300 versus 3 million cells of S. anatum) and species (S. anatum and S. heidelberg). Infection with either Salmonella species resulted in the development of a severe heterophilic leukopenia, and a significant increase in the percentage of both juveniles and lymphocytes by 48 hr postinoculation. Mortality rate was higher in groups of chicks inoculated with S. heidelberg than in groups given S. anatum.     

Effect of low and high doses ofSalmonella enteritidis PT4 on experimentally infected chicks

Chicks (1-d-old, three groups, each containing 50 chicks) were inoculated with 2×10² and 2×10⁸ CFU ofSalmonella enteritidis; the third group were kept as uninoculated control. Five birds from each group were euthanized at intervals from 6 h to 4 weeks post-inoculation (pi). In the lowdose groupS. enteritidis was isolated from 60% cecal samples at 18 h pi. and from 20% of livers at 3 d pi. Individual variation in the frequency ofS. enteritidis recovery was observed in this group. The clearance of salmonella from the organs was faster in the low-dose group, and salmonella was not isolated from the liver and cecum at 21 and at 27 d, pi, respectively. However, in the high-dose group,S. enteritidis was isolated from all ceca and 80% of liver 6 h pi, and salmonella was detected in the cecum and liver throughout the experiment. Serous typhlitis and unabsorbed yolk sac were the most prevalent lesions in both groups. Granulomatous nodules in the cecum were found occasionally in some cases in both inoculated groups, which can play a role as reservoirs in carrier chicks.     

A large panel of chicken cells are invaded in vivo by Salmonella Typhimurium even when depleted of all known invasion factors

Poultry are the main source of human infection by Salmonella. As infected poultry are asymptomatic, identifying infected poultry farms is difficult, thus controlling animal infections is of primary importance. As cell tropism is known to govern disease, our aim was therefore to identify infected host–cell types in the organs of chicks known to be involved in Salmonella infection and investigate the role of the three known invasion factors in this process (T3SS-1, Rck and PagN). Chicks were inoculated with wild-type or isogenic fluorescent Salmonella Typhimurium mutants via the intracoelomic route. Our results show that liver, spleen, gall bladder and aortic vessels could be foci of infection, and that phagocytic and non-phagocytic cells, including immune, epithelial and endothelial cells, are invaded in vivo in each organ. Moreover, a mutant defective for the T3SS-1, Rck and PagN remained able to colonize organs like the wild-type strain and invaded non-phagocytic cells in each organ studied. As the infection of the gall bladder had not previously been described in chicks, invasion of gall bladder cells was confirmed by immunohistochemistry and infection was shown to last several weeks after inoculation. Altogether, for the first time these findings provide insights into cell tropism of Salmonella in relevant organs involved in Salmonella infection in chicks and also demonstrate that the known invasion factors are not required for entry into these cell types.     

Metabolism of cholesterol in the tissues and blood of the chick embryo

Three artificially inseminated laying White Leghorn hens were given 35-50 micro c of cholesterol-4-(14)C intravenously. Their subsequently produced eggs contained cholesterol-(14)C-labeled yolks. Some of the fertilized eggs were analyzed for cholesterol content and radioactivity. Other eggs were incubated until hatching. The specific activity of the cholesterol contained in the serum and tissues of newly hatched chicks was determined and compared with that of yolk sac, which was taken as representative of egg yolk cholesterol before its metabolic transfer into the chick embryo. The specific activities of cholesterol in intestine, liver, serum, heart, and skeletal muscle and the whole chick were 95-98% of that in yolk sac, but that of brain cholesterol was only 11% of this value. These results indicate that whereas most of the cholesterol in the chick originated from the egg yolk, cholesterol biosynthesis was active in the brain and provided about 90% of its cholestero content. Newly hatched chicks were found to be hyperlipemic compared with older chicks and had fatty livers with a high cholesterol content. Desmosterol was found in 9- and 15-day old chick embryos but not in the newly hatched chicks, in which the only sterol was cholesterol.     

Infection and development of Nosema bombycis (Microsporida: Protozoa) in a cell line of Antheraea eucalypti

Spores of Nosema bombycis derived from diseased insects were highly purified by Urografin density gradient centrifugation. Antheraea eucalypti cells were inoculated with the purified spores primed with 0.1 n KOH solution to start a continuous propagation of N. bombycis in cell culture. The first increase in the number of infected A. eucalypti cells was observed at 48 hr postinoculation, and it was caused by the secondary infective forms of N. bombycis. The secondary infective forms were produced during the course of sporoblast differentiation. The parasites in cell cultures divided synchronously until 36 hr postinoculation. Mature spores were observed initially 6 days postinoculation at 27°C. The infected cultures were subcultured extensively for more than 1 year with the addition of healthy A. eucalypti cells.     

In ovo peptide YY and epidermal growth factor administration and their effects on growth and yolk utilization in neonatal meat-type chickens (Gallus domesticus)

The effects of in ovo peptide YY (PYY) or epidermal growth factor (EGF) administration on chick growth, yolk absorption and yolk stalk function in posthatch (0–5 days) meat-type or broiler chicks were determined. At Day 18 of incubation, treated eggs were injected into the air cell with 100 μl of either PYY (Trial 1) or EGF (Trial 2) at a dosage of 600 μg/kg egg weight. Saline-treated control eggs were injected similarly with 0.9% saline. At hatch, 200 μl of ⁵¹Cr-labeled microspheres were injected into chick yolk sacs. Epidermal growth factor increased ileal wet weight adjusted for body weight as well as ileal serosal dry matter. Body weight, feed consumption and excreta weight per bird, and relative weights of the yolk sac, intestine and liver were significantly affected by age of the chick in both trials. Relative radioactivity of the yolk sac, yolk stalk, blood, liver, and kidneys were affected by bird age in Trial 2; however, there were no significant effects due to PYY or EGF treatments on relative radioactivity of the tissues and organs examined. These data suggest that PYY and EGF had no effect on yolk absorption or yolk stalk function through 5 days in the posthatch chick.     

Experimental egg transmission of chicken anemia agent

When inoculated with chicken anemia agent (CAA) via the yolk sac at 6 days of age, chick embryos could develop normally into chicks. All the chicks hatched suffered from anemia and died at 10 to 15 days of age with bone marrow aplasia. Specific pathogen free laying hens were inoculated with CAA, and eggs were collected from them over a period from 1 to 28 days after inoculation. Two of 67 chicks hatched from the eggs revealed anemia at 14 days of age. CAA was recovered from 3 of 40 chicks. From the results, a possibility of egg transmission of CAA from dams to their progeny was experimentally suggested.     

Presence of sucrase in the yolk sac of the chick

The presence of sucrase in the yolk sac of the chick was studied biochemically and immunologically. The sucrase was partially purified from the yolk sac of hatched chicks and was compared with the sucrase purified from the small intestine. Immunodiffusion with antiserum against intestinal sucrase and characterization of the activity revealed that the two enzymes were almost identical. However, the size of the yolk sac sucrase was found to be slightly smaller than that of the intestinal enzyme by chromatography on Sephadex G-200 and polyacrylamide gel electrophoresis. Immunocytochemical studies showed that the sucrase was located on the free surface of yolk sac endodermal cells, but the sucrase may also be present in the cytoplasm.     

Pathogenicity of avian nephritis virus for embryonating hen's eggs

The pathogenicity of avian nephritis virus (ANV) for embryonating hen's eggs was studied by various routes of inoculation. When inoculated with ANV by the yolk sac route, 6-day-old embryos showed the highest susceptibility and all of them died 3 to 14 days postinoculation (PI). They manifested hemorrhage and edema of the whole body (3 to 6 days PI) and stunting (7 to 14 days PI). The 50% egg-infective dose of the virus by yolk sac inoculation coincided well with the virus titer expressed in plaque-forming units determined on the monolayer of chicken kidney cell cultures. The virus could be passed serially through the chorioallantoic membrane (CAM) of embryonating hen's eggs. In these eggs the CAM presented edematous thickening at the inoculation site, and the embryo stunting. when inoculated by the CAM route, high virus doses killed all embryos, but low virus doses allowed some of the infected embryos to hatch normally. When inoculated by the allantoic cavity route, the virus did not multiply in the allantoic cavity of embryonating eggs, but some of these eggs became infected. Fluorescent antigens were present only in the kidneys and CAM of embryos infected with the virus. The virus was recovered at a low rate from cloacal swabs of chicks from normally hatched eggs inoculated with the virus by the CAM route. These chicks were variable in growth, but had antibodies against the virus and developed nephritis at 36 days of age.     

Immunodiffusion test in avian encephalomyelitis. I. Standardization of procedure and detection of antigen in infected chickens and embryos.

The double immunodiffusion technique was applied to avian encephalomyelitis virus (AEV). Agar gel medium containing such a high concentration of NaCl as 15% was more preferable for highly diluted quantities of reactants than any other NaCl-containing medium. A single precipitin line appeared on the 1st to 7th days of diffusion at room temperature. The specificity of reaction between AEV antigen and homologous immune chicken serum has been demonstrated by no cross reaction between heterologous viruses and specific absorption by homologous virus. The antigen was produced in the brain, viscera, eyeball, whole body and yolk sac of chick embryos inoculated via yolk sac, as well as in the thigh muscles of chicks subcutaneously inoculated at 2 days of age. Antigenicity was detectable in 50% emulsion of these organs with a virus titer more than 10(5.0) per 0.1 g of tissue weight.     

Darkling Beetles (Alphitobius diaperinus) and Their Larvae as Potential Vectors for the Transfer of Campylobacter jejuni and Salmonella enterica Serovar Paratyphi B Variant Java between Successive Broiler Flocks

Broiler flocks often become infected with Campylobacter and Salmonella, and the exact contamination routes are still not fully understood. Insects like darkling beetles and their larvae may play a role in transfer of the pathogens between consecutive cycles. In this study, several groups of beetles and their larvae were artificially contaminated with a mixture of Salmonella enterica serovar Paratyphi B Variant Java and three C. jejuni strains and kept for different time intervals before they were fed to individually housed chicks. Most inoculated insects were positive for Salmonella and Campylobacter just before they were fed to the chicks. However, Campylobacter could not be isolated from insects that were kept for 1 week before they were used to mimic an empty week between rearing cycles. All broilers fed insects that were inoculated with pathogens on the day of feeding showed colonization with Campylobacter and Salmonella at levels of 50 to 100%. Transfer of both pathogens by groups of insects that were kept for 1 week before feeding to the chicks was also observed, but at lower levels. Naturally contaminated insects that were collected at a commercial broiler farm colonized broilers at low levels as well. In conclusion, the fact that Salmonella and Campylobacter can be transmitted via beetles and their larvae to flocks in successive rearing cycles indicates that there should be intensive control programs for exclusion of these insects from broiler houses.     

Sulfate esterifying activity of embryonic chick liver in vitro

A method has been described for the study of tissue sulfate-conjugating systems in vitro. Liver slices from embryonic chicks were maintained in vitro in a medium containing labeled inorganic sulfate and phenol. It was found that more of the sulfate was esterified at 20 °C. than at 37 °C. due to the longer continued activity at the lower temperature. All sulfate-esterifying activity was lost in liver slices maintained at 37 °C. for 30 hr. while those cultures maintained at 20 °C. continued to esterify sulfate for 70 hr.On the basis of our data there would appear to be a change in the thermal stability of the sulfate-esterifying enzyme system of the chick liver upon its transition from the embryonic stage to the stage of the fully developed chick. Data were presented for the chick 4 months ex ovo. We have been unable to detect any analogous temperature effects upon the sulfate-esterifying system in the livers of embryonic and adult rats.     

Verification of immunosuppression in chicks caused by Cryptosporidium baileyi infection using Brucella abortus strain 1119-3

Humoral immune response of young chicks to Brucella abortus strain 1119-3 inoculation was monitored to verify the degree of immunosuppression caused by infection with Cryptosporidium baileyi. Young chicks (2-day-old) were orally inoculated each with 2 × 10⁶ oocysts of C. baileyi, and then injected intramuscularly with 0.3 ml B. abortus strain 1119-3 containing 1 × 10⁹ living organisms on day 14 postinoculation (PI). Serum samples were tested by plate agglutination test on day 17 PI onwards at an interval of 3-6 days over a period of 36 days. Infected chicks with the coccidium showed significantly lower antibody titers than those of uninfected controls (P < 0.05). These findings document that C. baileyi infection in early life stage may predispose chicks easily to other potential poultry diseases.     

Plasmodium berghei: preparation of rat hepatic cell suspensions that include infective exoerythrocytic schizonts.

Employing an enzymatic method to dissociate rat liver, we prepared suspensions of liver cells from rats infected with sporozoites of Plasmodium berghei 3 to 10, 18 to 28, or 29 to 36 hr prior to liver dissociation. These suspensions of liver cells included hepatocytes, Kupffer cells, fibroblasts, and unidentified cells, as well as hepatocytes infected with exoerythrocytic schizonts (HEX) of P. berghei. These HEX were infective for recipient rodents when inoculated intraperitoneally into the recipients. The number of infective HEX present in the liver cell suspensions was quantitated by varying the number of HEX inoculated into recipients. This infectivity assay made it possible to compare the numbers of HEX in suspensions of liver cells from different donor rats. Infective HEX were obtained from donor rats in 35 of 41 experiments. The greatest number of infective HEX was obtained from donors injected with sporozoites 18 to 28 hr prior to liver dissociation. For morphological observation of mature HEX in cell suspensions, hepatic cells were prepared from donors infected with sporozoites 48 hr prior to liver dissociation. For experimental purposes, the preparation of infective HEX in suspensions of liver cells is superior to the preparation of infective HEX in liver fragments, because it is possible to quantitate the number of HEX which are present either visually or by means of the infectivity assay.     

Chlamydia trachomatis in cell culture. II. Susceptibility of seven established mammalian cell types in vitro. Adaptation of trachoma organisms to McCoy and BHK-21 cells

Trachoma organisms of serotype B were grown serially in irradiated cells (McCoy, BHK-21, Microbiological Associates, and BHK-21, Lister) and tested for infectivity in monolayers of five mammalian cell lines (BHK-21, CHO, HeLa S3, McCoy and OWMK) and two diploid strains (ST/BTL and WI-38). All cell types had low susceptibility to chlamydial infection but the number of inclusions increased when the inoculum was centrifuged onto the monolayers, or when the cells were irradiated. Infection was higher in non-irradiated CHO than in irradiated CHO in three out of a total of six experiments. Inclusion number was increased 300 times in HeLa S3 and up to three times in the other cell types after treatment with diethylaminoethyl-dextran (DEAE-D). Serial passage of Chlamydia trachomatis serotype B (strain Har-36) in CO60 McCoy and CO60 BHK-21 Lister resulted in partial adaptation of the strain to the host cell. The phenomenon of adaptation of serotype B to McCoy compensated for the lower susceptibility of this cell revealed when McCoy cells were inoculated with trachoma elementary bodies grown in BHK-21 Lister or in chick embryo yolk sac. Trachoma organisms of immunotypes A, B and C prepared in yolk sac produced more inclusion-forming units per ml in CO60 BHK-21 Lister than in CO60 McCoy.     

Leishmania in the Chick Embryo. II. Effects of Inoculum Size,Strain Origin,and Stage Injected and Infectivity of Embryo-Derived Parasites for Hamsters*

SYNOPSIS. Amastigotes of Leishmani donovani strains 2S, 3S, 3K, Hm, Gm, and Et were inoculated intravenously into 14-day chick embryos. The course of infection was followed by examinations of liver impression smears. With strain 33 at 33 C incubation, there was a 29-fold increase at 6 days postinfection when the inoculum contained ～4 × 10⁶ amastigotes, but only a ～6.3-fold increase when ～64 × 10⁶ parasites were injected. Infection courses of several geographic strains were compared at 30, 33, and 35 C incubation. Although the results were variable, Sudan strains 2S and 3S appeared to be separate isolations of a single strain. The Burma (Et), Kenya (3K), and Mediterranean (Hm, Gm) strains appeared to be distinct, but confirming evidence of their distinctness should be sought using serologic, epidemiologic, clinical, and biochemical criteria. Strains 2S and 3S multiplied best at 33 C or below, but the embryos usually failed to survive at 28 or 30 C. Multiplication was inhibited partially at 35 C and completely at 37 C. Inoculation of strain 3S promastigotes into chick embryos resulted in a loss of parasites in 1 hr to 2 days postinfection. Only amastigotes were seen in embryos incubated at 28 and 33 C for 4 days. Hamsters infected with parasites passaged once in chick embryos died at median postinoculation times that were closely comparable to those noted among hosts infected with amastigotes from hamster spleen.     

Destruction of Enteric Bacteria in Liquid Egg with β-Propiolactone

Liquid whole egg or egg white, inoculated with Escherichia coli 1485, Salmonella senftenberg ATCC 8400, or Salmonella typhimurium 84-I, was treated with concentrations of β-propiolactone ranging from 0.05 to 0.3%. Egg white containing 1 × 10³ to 1 × 10⁶ cells of E. coli 1485 per ml was sterilized in 1 hr at 27 C by lactone concentrations of 0.2 and 0.3%. Egg white containing 1 × 10⁵ cells of S. senftenberg ATCC 8400 per ml was sterilized in 12 hr at 10 C by 0.1% lactone and in 2 to 3 hr by 0.3% lactone at the same temperature.

Liquid whole egg inoculated with 1 × 10⁵ cells of either species of Salmonella was sterilized in 4 to 5 hr at 10 C with 0.2% lactone or in 2 to 3 hr by 0.3% lactone at this temperature. A mild heat treatment of either 15 min at 37 C or 1 min at 55 C markedly shortened the exposure times required for sterilization by β-propiolactone at 10 C.

After disinfection was complete, the lactone-treated liquid whole egg was reinoculated with low cell numbers of either species of Salmonella to determine the presence of residual lactone or toxic products. Liquid whole egg treated with 0.2% lactone would support the growth of salmonellae after 13 to 14 hr at 10 C. A heat treatment of 45 min at 37 C or 10 min at 55 C immediately after addition of 0.2% lactone allowed growth of the salmonellae in the lactone-treated liquid whole egg. No evidence of residual toxicity from the lactone treatment was found.

The amount of lactone needed to prevent the outgrowth of low cell numbers of either strain of Salmonella in liquid whole egg was quantitated. Liquid whole egg containing 0.06 to 0.07% lactone would not support salmonellae growth from inocula of 1 to 10 cells per ml of egg. Lactone concentrations above 0.08% prevented outgrowth of salmonellae inocula of 10 to 200 cells per ml of liquid whole egg.

     

Leishmania in the chick embryo: IV. Effects of embryo age and hatching,and behavior of L. donovani in cultures of chick fibroblasts

On incubation Days 9, 11, 12, 14, or 15, chick embryos were injected intravenously with 4.0 × 10⁶L. donovani amastigotes. Embryos were incubated at 33 C immediately after infection. Numbers of amastigotes found in the liver 1 hr after injection increased as the age of embryo recipients increased. Most 14- or 15-day infected embryos hatched when allowed to do so, but many younger embryos were unable to survive at 33 C. Numbers of amastigotes in the liver of chicks, hatched after infection as embryos, decreased as the cloacal temperature of the chicks increased. Despite a 31 C incubation temperature, chicks exhibited a mean 38.3 C cloacal temperature 1 day after hatching.Chick fibroblast cultures were initiated as explants of embryo brain and infected with amastigotes from hamster spleen. Only amastigotes were seen in cultures kept at 37 C, but extracellular promastigotes and intracellular amastigotes were present in cultures at 33 C. Although promastigotes increased in number in the medium overlay at 33 C, amastigotes decreased in number at 33 C and 37 C. One intracellular amastigote was seen in a culture which had been incubated at 25 C after inoculation with promastigotes.     

Maternal testosterone influences a begging component that makes fathers work harder in chick provisioning

In species with biparental care, parents disagree evolutionarily over the amount of care that each of them is willing to provide to offspring. It has recently been hypothesised that females may try to manipulate their mates by modifying offspring begging behaviour through yolk hormone deposition, shifting the division of labour in their own favour. To test this hypothesis we first investigated how yellow-legged gull (Larus michaellis) parents feed offspring in relation to each component of complex begging behaviour and if feeding behaviour varies between sexes. Then we investigated the effect of yolk testosterone on chicks' begging by experimentally increasing yolk testosterone levels. Our results revealed that yolk testosterone has a component-specific effect on chicks' begging, specifically increasing the number of chatter calls. Parental feeding effort was influenced by the number of chatter calls emitted by chicks, but most importantly, the influence was stronger in male than in female parents. Moreover, chick body mass increased with the number of paternal feeds. In conclusion, these results show that female gulls may use yolk testosterone deposition to exploit their partners as predicted by the ‘Manipulating Androgen Hypothesis (MAH)’.     

Physiological status of broiler chicks at pulling time and the relationship to duration of holding period

Newly hatched chicks may be held longer than 48 h and experience long periods of fasting in commercial hatcheries. Limited information is known about the physiological status of chicks in such situations, due to the difficulty of precisely recording time of hatch. This study investigated the effect of the time from hatch to pulling (holding period) on physiological measures/parameters in 109 broiler chicks. Fertile Ross 308 eggs were incubated in a custom built small-scale incubator. The individual hatching time of each focal chick was determined using eggshell temperature monitoring. At ‘pulling’ (512 h of incubation time), the quality of focal chicks was assessed using the chick scoring method and physiological parameters were measured including BW, organ (heart, liver and stomach) weights, blood values and plasma corticosterone level. The time from hatch to pulling varied from 7.58 to 44.97 h. Egg weight at setting was significantly correlated with chick BW and weight of organs at pulling, but had no effect on chick quality, blood values and plasma corticosterone. Relative BW at pulling was negatively associated with the duration of holding period (P=0.002). However, there was a positive correlation between relative stomach weight and the duration of the holding period (P<0.001). As the holding period duration increased, there was a trend that blood partial pressure of oxygen, haematocrit and haemoglobin also increased, and blood partial pressure of carbon dioxide, total carbon dioxide and bicarbonate decreased (P<0.05). A wide range of plasma corticosterone was observed from chicks that had experienced different durations of holding period. We conclude that shortening the hatch window and minimising the number of chicks that experience a long holding period before pulling may improve chick quality and physiological status, which may be due to unfavourable environmental conditions that include feed and water deprivation.