Infection of the Bovine Female Genital Tract with Chlamydia psittaci as a Possible Cause of Infertility

Contents: Chlamydia psittaci was isolated in embryonated chicken eggs via the yolk sac route from ten (16,7%) vaginal andlor endometrial mucosal scrapings of 60 slaughter cows. Simultaneous fecal shedding of chlamydiae was found in four animals. Chlamydial infections of the genital tract were frequent (p < 0,01) when there were endometrial inflammatory lesions together with the failure to detect other bacterial pathogens in the uterus.     

Diagnosis of avian chlamydiosis: specificity of the modified Giménez staining on smears and comparison of the sensitivity of isolation in eggs and three different cell cultures.

For the diagnosis of chlamydiosis in dead and live birds different methods were compared for their sensitivity and specificity. The specificity of the modified Giménez staining and the direct immunofluorescence (DIF) test for direct demonstration of Chlamydia psittaci in organ, cloacal and/or conjunctival smears was examined. The sensitivity of the isolation of Chlamydia psittaci in 6 days embryonated specific pathogen free (SPF) chicken eggs, Buffalo Green Monkey (BGM) cell line, McCoy cell line and Vero cell line was compared. On smears, the direct immunofluorescence test was more specific than the modified Giménez staining. The concordance between the results of both detection methods was 80%. The BGM cell culture was the most sensitive artificial host for isolation of Chlamydia psittaci, followed by the embryonated eggs, the Vero cell line and the McCoy cell line. The concordance between the results of isolation in BGM cell culture and eggs was 96.5%, while it was 86% between the results of isolation in BGM cell culture and Vero cell culture and only 65.5% between the results of isolation in BGM cell culture and McCoy cell culture. For dead bird species, chlamydiosis could be diagnosed more often using DIF on smears than with isolation. The concordance between the results of the DIF on smears and isolation followed by DIF was 91%.     

鹦鹉热嗜性衣原体感染SPF鸡和污染相关疫苗的初步调查

为初步调查SPF鸡感染鹦鹉热嗜性衣原体状况及相关SPF鸡胚源疫苗是否出现污染,本试验通过采集不同日龄的SPF鸡血清70份、SPF种蛋卵黄膜30份,收集市场上销售的SPF鸡胚源疫苗共41支,利用国产间接血凝试剂盒检测抗体,进口免疫荧光试剂盒分别测定其抗体、抗原阳性率,以评价SPF鸡鹦鹉热嗜性衣原体的流行状况和相关疫苗的污染状况。本试验结果显示,SPF鸡血清阳性率分别为31.4%(荧光法)、5.7%(间接血凝法);SPF种蛋阳性率33.3%,SPF鸡胚源疫苗平均阳性率31.7%。SPF鸡已经感染了鹦鹉热嗜性衣原体,且发现经鸡胚卵黄膜而传播病原的新途径,进而造成SPF鸡胚源疫苗出现衣原体污染。因此,加强SPF鸡鹦鹉热嗜性衣原体监测已势在必行。     

Experimental transmission of turkey viral hepatitis to day-old poults and identification of associated viral particles resembling picornaviruses.

Turkey viral hepatitis (TVH) was experimentally reproduced in two experiments in 1-day-old poults. In the first experiment, an infectious inoculum was prepared from filtered yolk materials harvested from dead embryonating chicken eggs (ECE) previously inoculated with suspensions of liver and pancreas tissues collected from TVH-affected birds in commercial turkey flocks. One-day-old poults given a yolk-sac inoculation or oral gavage with this preparation developed lesions in the liver and pancreas characteristic of TVH at 20 days postinoculation (PI) in 60% and 14% of the experimentally infected birds, respectively. With the identical inoculum, embryo mortality occurred at 8 and 10 days PI in embryonating turkey eggs (ETE) inoculated into the yolk sac. In the second experiment, an infectious inoculum was prepared from filtered yolk materials from dead ETE harvested in the first experiment. One-day-old poults given a yolk-sac inoculation with this filtered yolk material developed lesions in the liver and pancreas within 5 days PI. At 20 days PI, 67% of the experimentally infected birds had similar lesions. With the inoculum given to these poults, embryo mortality occurred at 6, 8, and 10 days PI in ETE inoculated into the yolk sac. Virus particles 26-28 nm in diameter with icosahedral morphology typical of picornaviruses were identified by EM in the yolk sacs of ETE that died in both experiments, and inoculated ETE that died following passage of filtered suspensions of pancreatic tissues collected from affected birds in the first experiment.     

Isolation and identification of Chlamydia psittaci from pet birds

Culturing of Chlamydia psittaci from pet birds requires the inoculation of a susceptible living host system with suspensions of various tissues from dead birds or with tracheal and/or cloacal swabs and fresh feces from live birds. Cell cultures have been used as the host system. The most commonly used cell cultures for isolation of C psittaci from pet birds are McCoy and mouse L cells. The sensitivity and specificity of cell culture equals or surpasses embryonating chicken eggs and mice, and results can be obtained in less than 7 days. To obtain satisfactory results, the inoculum must be centrifuged onto the cell cultures at 37 C, and the cells must be treated with a metabolic inhibitor such as colchicine or cycloheximide. Chlamydia psitaci can be detected in infected cells by use of fluorescent antibody, Giemsa, or Gimenez staining.     

Experimental infection of ducks with Mycoplasma gallisepticum

Specific-pathogen-free ducks 24 and 180 days old were inoculated intranasally with the S6 strain of Mycoplasma gallisepticum (MG). No significant gross lesions were found in trachea, lung or air sacs at 7 or 28 days postinfection (PI). MG was recovered from the infraorbital sinus and trachea but not from the air sacs 7 and 28 days PI. A few ducks responded serologically by developing agglutinating antibody. MG multiplied in embryonated duck eggs but to lower titers than in embryonated chicken eggs.     

Diagnosis of avian mycobacteriosis: comparison of culture,acid-fast stains,and polymerase chain reaction for the identification of Mycobacterium avium in experimentally inoculated Japanese quail (Coturnix coturnix japonica)

In this study we compared culture, acid-fast stains, and polymerase chain reaction (PCR) for the detection of acid-fast organisms in fecal and tissue samples from Japanese quail (Coturnix coturnix japonica) that were experimentally inoculated intravenously with Mycobacterium avium. For culture, three different culture media (modified Herrold egg yolk with mycobactin; Lowenstein-Jensen [L-J]; and L-J with cyclohexamide, naladixic acid, and lincomycin) were tested to determine which medium had the greatest success in isolating mycobacteria. Acid-fast staining methods included Zichl-Neelsen (Z-N) and Truant. The PCR assay detected mycobacterial DNA with primers specific for the 65-kD heat shock protein gene. Culture was considered the "gold standard." Compared with other culture media, L-J yielded more positive cultures and greater numbers of colonies on positive tubes, and incubation times were shorter. Mycobacterium avium was isolated from all of the harvested tissue samples (liver, spleen, and intestine) of inoculated birds. Mycobacteria were isolated from 53% (69/130) of fecal samples from inoculated birds. As the disease advanced, fecal culture was positive on more culture days, indicating that the culture-positive rate was higher later in the course of the disease. Compared with culture, all of the laboratory methods had 100% specificity for the tissue samples. Sensitivities for the tissue samples were 82.6% (Z-N), 95.7% (Truant), and 100% (PCR). For the fecal samples, the specificity was >95% for all methods. Sensitivities compared with fecal culture were 7.2% (Z-N), 30.4% (Truant), and 20.3% (PCR). Tissue and fecal samples from the two control birds were negative for acid-fast organisms by any method. These results were comparable with clinical cases of avian mycobacteriosis where culture and PCR of tissue samples seem to be the most sensitive and specific laboratory tests and evaluation of fecal samples still remains challenging. On the basis of the results of this study, identification of mycobacteria in fecal samples from Japanese quail can be optimized by repeated cultures and Truant acid-fast staining of fecal smears.     

Experimental infection of ducks with Mycoplasma synoviae

Specific-pathogen-free ducks 24 and 180 days old were inoculated intranasally with the WVU 1853 strain of Mycoplasma synoviae (MS). No significant gross lesions were found in the infraorbital sinus, trachea, or air sacs at 7 or 28 days postinfection (PI), although MS was recovered from all these organs. A few ducks responded serologically by developing agglutinating antibodies. MS multiplied in embryonated duck eggs but to lower titers than in embryonated chicken eggs.     

Characterisation of Chlamydia psittaci isolated from a horse

This paper describes the isolation and characterisation of a strain of Chlamydia psittaci obtained from a nasal swab taken from a horse with serous nasal discharge. Initial isolation was achieved in cycloheximide-treated McCoy cell monolayers. Chlamydial inclusions stained by immunofluorescence either with a rabbit antiserum raised against C. psittaci or with a monoclonal antibody directed against the genus-specific lipopolysaccharide antigen were single and compact. They did not stain with iodine or with a monoclonal antibody reactive against Chlamydia trachomatis. The agent was re-isolated in the yolk sacs of embryonated hens eggs and designated N16. Identification of the agent was confirmed by electron microscopy. Unique plasmid DNA was prepared from a purified suspension of chlamydial elementary bodies (EBs), and analysed by electrophoresis through 1.0% agarose gels stained by ethidium bromide. This strain of C. psittaci grew relatively slowly in cycloheximide-treated McCoy cells, and the yield of elementary bodies during the course of one growth cycle was relatively low.     

Methods of detection of Chlamydia psittaci in domesticated and wild birds

OBJECTIVE: To study the occurrence of Chlamydia psittaci in domesticated and wild birds and compare the sensitivity of molecular detection with cell culture isolation. DESIGN: Study of cell culture isolation and PCR detection of C psittaci in avian samples. PROCEDURE: Samples were obtained from 485 birds. Domesticated birds were selected at random from pet shops, private aviaries and zoos, while wild birds were captured locally, sampled, and immediately released. Swabs were collected from choanal slit, conjunctiva and cloaca of each bird and pooled. Samples were divided into equal portions for use in PCR dot-blot and cell culture detection. PCR and dot-blot detection was based on the ompB gene. RESULTS: Prevalence of infection varied markedly between flocks of captive birds. It was highest where there were frequent changes in the flock members or where there were many birds confined in small areas. C psittaci was not detected in wild birds or water birds. The sensitivity of cell culture compared to PCR dot-blot detection was 68%. All samples positive by cell culture were also positive by PCR. CONCLUSIONS: PCR-dot blot detection of C psittaci in birds appears to be more sensitive than cell culture isolation in this study. C psittaci infection of birds may occur in clinically normal captive birds.     

火鸡冠状病毒的分离和初步鉴定

2003年国内某火鸡场发生了一种以侵害15～25日龄雏火鸡为主的急性传染病，主要表现为腹泻，十二指肠、直肠充血和出血，盲肠肿大，肠道内充满黄绿色内容物，死亡率约为10％～20％。取病死火鸡肝、脾、肠匀浆，取上清液通过尿囊腔接种15日龄SPF鸡胚。连续传代至第5代，收集接种后72h内死亡或存活鸡胚的卵黄和肠道，用于病毒分离和提纯。试验中发现该病毒能凝集兔红细胞，不能凝集鸡红细胞。经电镜观察，在病毒提纯液中发现有圆形或椭圆形、带花冠状纤突的病毒粒子，初步诊断为火鸡冠状病毒感染。进而设计针对火鸡冠状病毒S2基因引物，进行RT-PCR扩增，结果扩增出预期大小的片段。运用所分离病毒进行动物回归试验，感染火鸡出现与自然病例一致的临床症状和病理变化，并能从发病火鸡分离出该病毒。以上结果表明所分离的病毒为火鸡冠状病毒。此病毒的分离在国内尚属首例。     

Chlamydiae and atherosclerosis: can psittacine cases support the link?

Atherosclerosis is a common disease in pet birds, particularly in psittacines. Little is known about the role of risk factors predisposing birds to this disease. In our study, we tried to detect chlamydiae in formalin-fixed and paraffin-embedded atherosclerotic tissue from 103 pet birds to clarify their role in atherosclerosis. Methods used were polymerase chain reaction (PCR), sequencing, and immunohistochemistry. Histopathologic examination served to classify the extent of atherosclerotic lesions. In the PCR, 4 (3.9%) of 103 cases, all of them with advanced stages of atherosclerosis, were positive. Subsequent sequence analysis revealed high identities (94%-100%) with Chlamydophila psittaci in three cases. Interestingly, two of these birds came from C. psittaci-infected populations. Because of the low incidence (3.9%), the occurrence only in advanced stages, and the association with C psittaci-infected avian populations, a causal relationship between chlamydiae and atherosclerosis in pet birds is rather improbable.     

Isolation of Chlamydia psittaci from bull ejaculate

During the repeated serological examination (RVK) in five breeding bulls the positive levels of antibodies to Chlamydia psittaci in titre 1 : 128 were found. In the isolation experiments the pelleted ejaculates deposited in liquid nitrogen were used. The isolation of Chlamydia psittaci on yolk sacs of chicken embryos was positive in two breeding bulls. The isolated strains are labelled GN-33 and OK-107. The serological examination of blood samples was in all five breeding bulls negative on brucellosis (BAB), infectious bovine rhinotracheitis and coxiellosis and positive on PI-3. Bacteriological examination of spermatic fluid proved only sporadic contamination with moulds and saprophytic bacteria.     

Isolation and characterization of peacock Chlamydophila psittaci infection in China

The objective of this study was to isolate and identify suspected pathogens from peacocks and peacock farmers with severe pneumonia and to investigate its potential association with peacocks' pneumonia, caused by Chlamydophila psittaci infection. A clinical examination of infected peacocks identified birds with symptoms of anorexia, weight loss, yellowish droppings, airsacculitis, sinusitis, and conjunctivitis, whereas the infected farmers showed high fever and respiratory distress. Immunofluorescence tests detected chlamydial antigens in pharyngeal swabs (12 of 20) and lung tissue samples (four of five) from peacocks. One of four swabs taken from farmers was also positive by the same test. Specific anti-chlamydia immunoglobulin G was detected in 16 of 20 peacocks and four of four peacock farmers. The isolated pathogen was able to grow in specific-pathogen-free (SPF) chicken embryos and McCoy cell lines and was identified as Chlamydiae by immunofluorescence assay and PCR. Avian influenza virus, Newcastle disease virus, and infectious bronchitis virus were eliminated as potential causative agents after pharyngeal swabs inoculated onto the chorioallantoic membrane of embryonate eggs failed to recover viable virus. PCR and restriction fragment length polymorphism indicated the ompA gene from the isolate was similar to that of avian C. psittaci type B. Three-week-old SPF chickens challenged with the peacock isolate via intraperitoneal injection showed a typical pneumonia, airsacculitis, and splenitis. Subsequently, the inoculating strain was recovered from the lungs of challenged birds. This is the first report of C. psittaci infection in peacocks and peacock farmers.     

Fetotoxicity of some anticoccidial drugs in chickens

Fetotoxic effects induced by three anticoccidial drugs: robenidine, salinomycin and arprinocid were elucidated in the chicken. Different doses of these drugs were inoculated in groups of embryonated chicken eggs by the yolk sac route. After inoculation, candling of the eggs was performed daily and embryonic or fetal mortalities were recorded. At 19 days old, alive fetuses were collected, weighed, measured and examined morphologically for abnormalities. A group of eggs was kept non-inoculated as a control and another was inoculated with the solvent of the tested drugs. Inoculation of 0.09-9.75 mg robenidine/egg, 0.06-6.75 mg salinomycin/egg or 0.08-8.25 mg arprinocid/egg into the yolk sac of 7 days old embryos caused a dose-dependent fetal death. Arprinocid was the most lethal to chicken fetuses, followed by salinomycin while robenidine was the least. Dead fetuses were usually haemorrhagic, dwarfish and friable. Surviving fetuses showed a dose-dependent reduction in body weight and length, insignificant decrease in leg and wing lengths as well as some developmental abnormalities.     

Differences in the induction of urate deposition of specific-pathogen-free chicks inoculated with avian nephritis virus passaged by five different methods.

The G-4260 strain of avian nephritis virus (ANV) was passaged using five different methods as follows: method 1, passage three times in chorioallantoic membrane (CAM) of 11-day-old embryonated eggs (CAM3); method 2, passage twice in CAM and further passage once in yolk sac (YS) of 6-day-old embryonated eggs (CAM2-YS1); method 3, passage 11 times in CAM (CAM11); method 4, passage 10 times in CAM and further passage twice in YS (CAM10-YS2); method 5, passage as in Method 4 and then passage three times in chicken kidney cell culture (CAM10-YS2-CK3). CAM11 and CAM10-YS2 were each inoculated orally into 25 one-day-old specific-pathogen-free (SPF) chicks. Seven chicks in the CAM11-inoculated group and six chicks in the CAM10-YS2-inoculated group died or were killed because they were moribund; all had either nephrosis or urate deposition. CAM3, CAM2-YS1, CAM10-YS2, and CAM10-YS2-CK3 were each inoculated intraperitoneally into 15 one-day-old SPF chicks. No chicks inoculated with CAM3 or CAM2-YS1 died, but wo chicks inoculated with CAM10-YS2 and three inoculated with CAM10-YS2-CK3 died with urate deposition. At 14 postinoculation, plasma urate values of the CAM10-YS2- and CAM10-YS2-CK3-inoculated chicks were significantly higher than those of CAM3- and CAM2-YS1-inoculated chicks and control chicks (P less than 0.01). However, interstitial nephritis was observed in most of the ANV-inoculated birds.     

Pathogenesis of Salmonella enteritidis infection in laying chickens. I. Studies on egg transmission, clinical signs, fecal shedding, and serologic responses

Laying hens were inoculated orally, intracloacally (IC), or intravenously (IV) with Salmonella enteritidis phage type 8 isolates from a human (E700-87) eggs (Y-8P2), or the ovary of a hen (27A). Oral or IV inoculation of 2 x 10(8) to 4 x 10(8) colony-forming units (CFU) of E700-87 caused depression, anorexia, reduced egg production, diarrhea, and some mortality. Lower doses resulted in milder clinical signs. S. enteritidis was cultured from the shells of a few eggs but not from egg contents. Fecal shedding persisted for up to 6 weeks in some birds. Isolate Y-8P2 (10(6) CFU) also caused anorexia, diarrhea, and a drop in egg production. Hens inoculated orally or IC were less severely affected than those inoculated IV. Fecal shedding was intermittent and lasted up to 18 days. Eggshells from the IC-inoculated birds had the highest rate of contamination, and S. enteritidis was isolated from the albumen of 11 and yolk of three of 726 eggs. Oral inoculation of 10(6) CFU of isolate 27A resulted in a bacteremic infection with seeding of the liver, spleen, peritoneum, ovule, and oviduct. However, the birds remained clinically normal with normal egg production. S. enteritidis was cultured from the yolk and albumen of a small number of eggs until 11 days postinfection. Antigen prepared from S. enteritidis detected antibody in more sera than did commercially available S. pullorum antigen in agglutination tests.     

Clinical effects, virological and serological responses in chickens following in-ovo inoculation of reticuloendotheliosis virus

Six-day-old embryonated specific pathogen free chicken eggs were inoculated with reticuloendotheliosis virus (REV) into the yolk sac and were incubated until they hatched. The hatchability of eggs inoculated with REV was significantly less (P less than 0.025) than that of media-inoculated controls. Although there were no significant differences in the body weights of these chickens at hatching, there were differences (P less than 0.001) at 6, 25 and 51 days of age between the infected and control chickens. Six of 10 chickens hatched from eggs inoculated with REV had feathering defects at 6 days of age. All chickens hatched from infected eggs had cell-free viraemia and antigenaemia, but not precipitating antibodies. Some of these chickens had very low neutralising antibody titres (less than 45) when examined at 25 and 37 days of age, as did all 10 chickens at 51 days of age. A low rate of horizontal transmission was indicated by the detection of antibodies at 37 and 51 days of age in chickens running in contact with the chickens hatched from eggs inoculated with REV.     

Isolation of Chlamydia psittaci and Moraxella bovis from infectious keratoconjunctivitis in lambs

Out of 189 lambs in the flock, 25 animals suffered from bilateral or unilateral conjunctivitis, or keratoconjunctivitis. By serological examination (RVK), positive levels of antibodies to the group-specific antigen of Chl. psittaci were found in three out of six lambs examined by laboratory methods. Bacteriological examination of eye smears of six lambs showed in four cases the infection by microorganisms of Moraxella bovis. Smears from the conjunctivas of these lambs were after preparation instilled in the yolk sacs of six to seven days old chicken embryos. One strain of Chlamydia psittaci was isolated from the same material as Moraxella bovis.     

Response of two breeds of chickens to Ascaridia galli infections from two geographic sources

Comparative resistance to different isolates of Ascaridia galli was investigated in a local chicken breed from Jordan (LC) and in the Lohmann LSL white chicken (LW) strain. In two trials, birds of LC and LW were inoculated orally at 1-day old with 250 embryonated A. galli eggs. In the first trial a German source of A. galli was used, whereas in the second trial, a Jordan source of A. galli was used. At week 7 of infection, infected LC birds harbored significantly (P<0.05) fewer worms and excreted less A. galli eggs than infected LW birds. A. galli isolated from Jordan were less infectious than A. galli from Germany. Results suggest that the variation in genetic background between LC and LW is involved in the resistance to A. galli infection. A. galli isolates from different geographic areas differ in their ability to infect different chicken genotypes.