Pathogenicity and antigenicity of avian nephritis isolates.

The G-4260, IR-N, M-6, and M-8 strains of avian nephritis virus (ANV) were inoculated orally into 1-day-old specific-pathogen-free chicks of the line PDL-1 for pathological and serological study. Five of 15 chicks inoculated with the G-4260 strain died with visceral urate depositions. One of 15 chicks each inoculated with the M-6 and M-8 strains died with nephrosis and visceral urate deposition, respectively. No chicks inoculated with the IR-N strain died. Mean body weights of ANV-inoculated chicks, except for the IR-N-inoculated chicks, at 14 days postinoculation (PI) were significantly lower than those of control chicks (P less than 0.01). However, interstitial nephritis was observed in all ANV-inoculated birds that were histopathologically examined at 14 days PI. In the serological study, the G-4260 and IR-N strains were classified as the same serotype, and the M-6 and M-8 strains were classified as a different serotype from the G-4260 and IR-N strains. These results indicate that there at least two serotypes of ANV and its strains differ in pathogenicity.     

Pathologically and serologically different avian nephritis virus isolates implicated in etiology of baby chick nephropathy.

Three virus isolates (WG-3, -4, and -5) from chicks affected by baby chick nephropathy were orally inoculated into 1-day-old specific-pathogen-free chicks of lines PDL-1 and 15I. Additional chicks were orally inoculated with avian nephritis virus (ANV) strain G-4260. Chicks inoculated with isolates WG-3, -4, and -5 died between 2 and 6 days postinoculation (PI), with mortality ranging from 0% to 53.3%. Pathological findings in the dead chicks included nephrosis in chicks inoculated with WG-3, -4, and -5, and nephritis and visceral urate deposition in chicks inoculated with G-4260. The stability of the WG-5 isolate, as well as the size of the particles and the nucleic acid type, were also similar to those of the G-4260 strain. All of the examined chicks inoculated with WG-3, -4, and -5 had interstitial nephritis at 14 days PI. Therefore, the three virus isolates were considered to be ANV. However, there was no serological relationship between the isolates and ANV (G-4260 and M-8 strains).     

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.     

Visceral urate deposits in chicks inoculated with avian nephritis virus

Avian nephritis virus, G-4260 strain, was inoculated orally into one-day-old specific-pathogen-free chicks of two lines. Approximately 20 per cent of the chicks of both lines died with visceral urate deposits from eight to 12 days after infection, and the virus was isolated from the kidneys of the dead chicks. At 14 or 15 days of age the mean liveweight of the surviving infected chicks was approximately 16 per cent less than that of the uninfected control chicks.     

Enteropathogenicity of Dutch and German avian reoviruses in SPF white leghorn chickens and broilers

The enteropathogenicity of avian reoviruses (ARVs), isolated from chickens affected with malabsorption syndrome (MAS) from The Netherlands and Germany was studied. In the first trial seven different ARVs isolated from MAS cases were inoculated in 1-day-old specific pathogenic free (SPF) white leghorns. The pathogenicity was compared with 2 ARVs isolated from cases of tenosynovitis, namely reference strain S1133 and a Dutch strain. Although a difference in the severity of the clinical disease was observed, all reoviruses could induce vacuolar degeneration and sloughing of the epithelium of the small intestine at day 2 post inoculation (PI) till day 7 PI. Two Dutch and one German ARV derived from MAS causing the most severe intestinal lesions at day 2 PI, were further studied in the second trial using SPF broilers. These reoviruses did not cause weight gain depression in the broilers although lesions in the small intestine were present from day 1 up to day 4 PI and were more severe than in the white leghorn chickens. In one of the inoculated groups apical denuded villi were already present at day 1 PI. At day 7 PI the small intestine of the infected broilers appeared to be normal. Reovirus antigen was detected in the cytoplasm of the enterocytes at the tip and middle section of the affected villi both in layers and in broilers. To study the role of intestinal CD4+ and CD8+ T-cells and macrophages/monocytes in the pathogenesis of ARV, the numbers of these cells of the jejunal villi of one infected and the control broiler groups were compared. CD4+ T-cells were detected in low numbers and only in the infected broiler group at day 14 PI. The numbers of CD8+ T-cells and macrophages/monocytes were significantly higher in the infected broiler group than in the control broiler group at day 7 and 14 PI and at day 7 PI respectively. Our study indicates that the reovirus alone cannot induce intestinal lesions as found in MAS chickens. Moreover, CD8+ T-cells may play a major role in the pathogenesis and or reovirus clearance in the small intestine.     

Experimental factors affecting mortality following inoculation of chickens with avian nephritis virus (G-4260).

Groups of approximately 20 one-day-old chickens were inoculated with G-4260, the reference strain of avian nephritis virus (ANV), or saline. Based on mortality rates from severe nephritis in comparable experiments, light Sussex chickens generally were more susceptible than Rhode Island red (RIR) chickens. Mortality was greater in those given broiler starter than those given other feeds, and was greater when light Sussex chickens were given broiler starter feed and cold-stressed at 15 +/- 1 C for 2 hr daily during the first week rather than brooded normally. Inoculation with G-4260 either orally or by intraperitoneal injection produced similar results in RIR chickens. Thirty-three inoculated chickens died of severe nephritis between 4 and 12 days postinoculation, and 24 (73%) of them had visceral urate deposits. Inoculated inbred white leghorn Line 15 chickens with maternal antibody to ANV were brooded normally and given broiler feed: they were susceptible to infection as evidenced by subsequent histological lesions in the kidneys and serology, but mortality was not a feature. There were no deaths from nephritis in inoculated non-inbred white leghorn chickens free of maternal antibody to ANV that were given broiler feed and brooded normally. These results have implications in standardizing experimental conditions for the study of mortality induced by G-4260 and similar viruses.     

Early pathogenesis in chicks of infection with an enterotropic strain of infectious bronchitis virus

One-day-old specific-pathogen-free chicks were inoculated intranasally and intraocularly with infectious bronchitis virus (strain G). At days 1, 3, 5, 7, 10, and 14 postinfection, three birds were euthanatized, and the virus contents of both enteric tissues and some non-enteric tissues were assayed. Immunofluorescence and histopathological studies were also conducted. Six of 30 chicks died of nephritis between days 5-10 postinfection. Gross kidney lesions were the major pathological abnormalities. Inflammation was observed histologically in trachea, kidney, and rectum. High virus titers were found at various times in trachea, kidney, and all enteric tissues except for the jejunum. Relatively high titers of virus were still detectable at day 14 postinfection in the kidney, proventriculus, cecal tonsil, ileum, rectum, and bursa of Fabricius. Immunofluorescence staining showed viral antigens in enterocytes at the tips of villi in the ileum and rectum, and in the bursa. Viral antigens were also demonstrated in the epithelial cells of the trachea and in kidney tubules.     

Detection of avian nephritis virus in Australian chicken flocks

Avian nephritis virus (ANV) is thought to infect poultry flocks worldwide, but no confirmed case has been reported in Australia. The first such case is described in this study. Cases of young chickens with clinical signs of dehydration and diarrhea were submitted to our laboratory and histopathology detected interstitial nephritis. Vaccine strains of infectious bronchitis virus were detected in some of these cases but were not considered to be the causative agent. A total of seven fresh submissions from broiler chicken flocks were collected at 8-11 days of age. Degenerate PCR primers were designed based on published ANV polymerase gene sequences and used to analyze historic cases as well as the fresh submissions. Six of the seven fresh submissions, and one historic case, were positive for ANV with nucleotide sequencing confirming these results. These results establish ANV as an infectious pathogen circulating in Australian poultry.     

Experimental avian paramyxovirus serotype-3 infection in chickens and turkeys

Avian paramyxoviruses (APMV) are divided into nine serotypes. Newcastle disease virus (APMV-1) is the most extensively characterized, while relatively little information is available for the other APMV serotypes. In the present study, we examined the pathogenicity of two divergent strains of APMV-3, Netherlands and Wisconsin, in (i) 9-day-old embryonated chicken eggs, (ii) 1-day-old specific pathogen free (SPF) chicks and turkeys, and (iii) 2-week-old SPF chickens and turkeys. The mean death time in 9-day-old embryonated chicken eggs was 112 h for APMV-3 strain Netherlands and > 168 h for strain Wisconsin. The intracerebral pathogenicity index in 1-day-old chicks for strain Netherlands was 0.39 and for strain Wisconsin was zero. Thus, both strains are lentogenic. Both the strains replicated well in brain tissue when inoculated intracerebrally in 1-day-old SPF chicks, but without causing death. Mild respiratory disease signs were observed in 1-day-old chickens and turkeys when inoculated through oculonasal route with either strain. There were no overt signs of illness in 2-weeks-old chickens and turkeys by either strain, although all the birds seroconverted after infection. The viruses were isolated predominantly from brain, lungs, spleens, trachea, pancreas and kidney. Immunohistochemistry studies also showed the presence of large amount of viral antigens in both epithelial and sub-epithelial lining of respiratory and alimentary tracts. Our result suggests systemic spread of APMV-3 even though the viral fusion glycoprotein does not contain the canonical furin proteases cleavage site. Furthermore, there was little or no disease despite systemic viral spread and abundant viral replication in all the tissues tested.     

The effect of Eimeria acervulina infection on hearts of young broiler chicks

Hearts from 2-to-3-week-old broiler chicks that were inoculated with 2 X 10(6) sporulated oocysts/bird weighed significantly less than hearts from control chicks at 4 and 7 days postinoculation (PI). However, heart weights of infected chicks expressed as percentage of body weights were not significantly different from those of controls throughout the experiment. Infection also had no significant effect on heart protein content. Cardiac glycogen was significantly increased at 7 days PI but not at 4 days PI. Oxidation of both alpha-ketoglutaric and octanoic acids by mitochondrial preparations from hearts of infected chicks was significantly depressed at 4 days PI compared with controls; at 7 days PI, however, oxidation of octanoic acid was significantly increased, but that of alpha-ketoglutaric acid was not.     

A paramyxovirus of serotype 3 isolated from African and Australian finches

Two virus isolates were obtained from exotic finches (Ortygospiza atricollis and Poephila cincta) suffering from apathy, diarrhea, conjunctivitis, and dysphagia. The isolates were identified as paramyxoviruses based on their multiplication characteristics in embryonating chicken eggs, chicken embryo fibroblasts, and chicken embryo kidney cell cultures, on morphology upon electron microscopy, and on other biological properties. Both isolates were serologically related to the reference strain of the paramyxovirus serotype 3. Intravenous infection of 42-day-old chicks resulted in no clinical signs, but intracerebral infection of 1-day-old chicks resulted in mortality and intracerebral pathogenicity indices of 0.25 to 0.35. Of five finches from various species inoculated with isolate 840/85, three remained clinically healthy through 6 weeks, but two died: one (Poephila cincta) 5 days postinoculation after showing nervous distress, and the other (Amandava amandava) suddenly 42 days postinoculation.     

Pathogenesis of two strains of avian paramyxovirus serotype 2, Yucaipa and Bangor, in chickens and turkeys

Nine serologic types of avian paramyxovirus (APMV) have been recognized. Newcastle disease virus (APMV-1) is the most extensively characterized virus, while relatively little information is available for the other APMV serotypes. In the present study, we examined the pathogenicity of two strains of APMV-2, Yucaipa and Bangor, in 9-day-old embryonated chicken eggs, 1-day-old specific-pathogen-free (SPF) chicks, and 4-wk-old SPF chickens and turkeys. The mean death time in 9-day-old embryonated chicken eggs was more than 168 hr for both strains, and their intracerebral pathogenicity index (ICPI) was zero, indicating that these viruses are nonpathogenic in chickens. When inoculated intracerebrally in 1-day-old chicks, neither strain caused disease or replicated detectably in the brain. This suggests that the zero ICPI value of APMV-2 reflects the inability of the virus to grow in neural cells. Groups of twelve 4-wk-old SPF chickens and turkeys were inoculated oculonasally with either strain, and three birds per group were euthanatized on days 2, 4, 6, and 14 postinoculation for analysis. There were no overt clinical signs of illnesses, although all birds seroconverted by day 6. The viruses were isolated predominantly from the respiratory and alimentary tracts. Immunohistochemistry studies also showed the presence of a large amount of viral antigens in epithelial linings of respiratory and alimentary tracts. There also was evidence of systemic spread even though the cleavage site of the viral fusion glycoprotein does not contain the canonical furin protease cleavage site.     

The distribution of avian enteroviruses in Germany]

Sera from chicken flocks of different regions in Germany were examined for antibodies to avian nephritis virus (ANV) by indirect immunofluorescence. There was no evidence of a wide distribution of ANV, at least in chicken parent flocks, in contrast to results from investigations in England, Northern-Ireland and Japan.     

Colonisation studies using Campylobacter jejuni in chicks

White Leghorn chicks used in this study were hatched from specific pathogen-free eggs. The colonizing capability of Campylobacter (C.) jejuni strains was investigated in 6 experiments. The formation of specific antibodies associated to colonization was also detected. In each experiment, day of hatch chicks were randomly separated into three groups of 24 birds each: two groups colonized experimentally and one control group. Chicks were reared on the floor in three separated, adjacent rooms with sterilized wood shavings as litter. At 2 or 8 days of age, respectively, the chicks in the experimentally colonized groups received between 3.3 x 10(7) and 2.0 x 10(8) colony-forming units (CFU) of C. jejuni via oesophageal gavage. Furthermore, 7, 14, 21, 28, 42 and 56 days after inoculation, 4 chicks of each group were sacrificed by cervical dislocation, at which time blood, liver and faeces were collected for processing. Serum was centrifuged and Campylobacter-specific IgG, IgA and IgM antibodies were measured by an indirect enzyme-linked immunosorbent assay (ELISA). Altogether, the colonizing capability of 11 C. jejuni strains was examined. Surprisingly, there were large differences between the C. jejuni isolates. After these experiments, we could divide the isolates into three groups. 4 out of 11 isolates could not be reisolated, 2 isolates caused weak or delayed colonization and 5 C. jejuni produced strong, long-lasting colonization. In the first days of life (9 days), the C. jejuni-free SPF chicks (control animals) had high IgG titres in sera, which decreased markedly up to the age of 15 days. During the experiments the IgM and IgA titres remained nearly at the same level, i.e., the amounts of maternal antibodies were low and there was no evidence for antibody formation in the chicks themselves. Two- and 8-day-old chicks were inoculated with C. jejuni strain Penner 1. Two-day-old chicks were colonized 3 weeks after inoculation. In comparison with these animals, 8-day-old chicks were colonized already 2 weeks after inoculation. There is the assumption, that the higher maternal antibodies in 2-day-old chicks could be responsible for this delay. In chicks the C. jejuni colonization resulted in a marked IgG (but not IgM and IgA) increase. Apparently, there is a positive relationship between the counts of this pathogen in caeca and the IgG increase.     

Consequences to chicks hatched from Escherichia coli-inoculated embryos.

An Escherichia coli causing negligible mortality in embryonated chicken eggs was adapted to grow in media containing nalidixic acid. This isolate (EcNAL) was inoculated into 12-day-old embryonated eggs. Additional embryos inoculated with tryptose phosphate broth (TPB) served as controls. Six days later, all surviving eggs were moved to hatching units. One hatcher contained half of the TPB-inoculated eggs; the chicks hatching from these eggs served as negative controls. The EcNAL-inoculated eggs and the remaining TPB-inoculated eggs were moved to a second hatcher and allowed to hatch together; chicks hatching from these TPB-inoculated eggs served as contact controls. On day of hatch and at intervals thereafter, chicks from each of the treatment groups were sampled. Their body and yolk weights were recorded, and various tissues were cultured for the presence of the EcNAL bacterium. Hatchability of the EcNAL-inoculated embryos was markedly lower than that of either control group. Chicks from EcNAL-inoculated embryos also had low but detectable levels of mortality, lowered body weights, and increased yolk-to-body weight ratios. These same chicks had persistently high levels of EcNAL in the yolk and lower but detectable levels of the organism in the lungs and tracheas, which lasted a few days. The contact controls, on the other hand, were similar to the negative controls as far as having negligible mortality, steadily increasing body weights, and declining yolk-to-body weight ratios. However, in contrast to the negative controls, EcNAL was recovered primarily from the respiratory tract of the contact controls for a brief period of 3-4 days after hatch.     

A sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: immunostaining of cryostat sections and virus isolation

The sequential tissue distribution of virus was investigated using virus isolation and immunofluorescence tests in 1-day-old piglets inoculated with porcine circovirus 2 (PCV2) and/or porcine parvovirus (PPV). Enlarged mesenteric lymph nodes were seen in the pig inoculated with PCV2 alone and killed at 26 days post-inoculation (PI). One of the pigs inoculated with PCV2 and PPV and killed at 21 days PI had an enlarged liver. The pig killed at 26 days PI in this group had enlarged liver, kidneys and heart. Histopathological changes were seen in lymphoid tissues of the pigs inoculated with PCV2 alone and killed at 14 and 26 days PI. Similar, but more severe, lesions were observed in the pigs infected with PCV2 and PPV and killed from 10 days PI onwards. Histological lesions of nephritis, pneumonia and hepatitis were also apparent in these animals. Mild nephritis was also seen in the pigs infected with PPV alone and killed at 14 and 26 days PI. Moderate amounts of PPV antigen were detected in tissues from the pigs inoculated with PPV alone and killed at 14 days PI. Low levels of PCV antigen were detected, mainly in lymphoid tissues, in the pigs inoculated with PCV alone and killed at 14 days PI. Low to moderate amounts of PCV antigen were detected in a wider range of tissues in the pig in this group killed at 26 days PI. In the pigs inoculated with both viruses, PPV antigen was detected in tissues of pigs killed from 3 to 26 days PI with maximal amounts detected between 6 and 14 days PI. PCV2 antigen was detected in low to moderate amounts in the tissues of pigs killed at 14 days PI. Large amounts of PCV2 antigen were detected in most of the tissues from pigs in this group killed between 17 and 26 days PI. Virus isolation results for PCV2 generally correlated well with the results for immunofluorescent staining. PPV was isolated from almost all tissues from pigs inoculated with PCV2 and PPV, a much higher incidence of positive tissues than observed for immunofluorescent staining.     

Pathogenicity of different serogroups of avian salmonellae in specific-pathogen-free chickens.

The pathogenicity of one isolate of Salmonella typhimurium, four isolates of Salmonella heidelberg, three isolates of Salmonella kentucky, two isolates of Salmonella montevideo, one isolate of Salmonella hadar, and two isolates of Salmonella enteritidis (SE), one belonging to phage type (PT)13a and the other to PT34, was investigated in specific-pathogen-free chicks. Three hundred eighty-four chicks were separated into 16 equal groups of 24 chicks. Thirteen groups were inoculated individually with 0.5 ml of broth culture containing 1 x 10(7) colony-forming units (CFU) of either S. typhimurium (one source), S. heidelberg (four sources), S. montevideo (two sources), S. hadar (one source), S. kentucky (three sources), SE PT 13a (one source) or SE PT 34 (one source) by crop gavage. Two groups of 24 chicks were inoculated in the same way with 1 x 10(7) CFU of SE PT4 (chicken-CA) and Salmonella pullorum. Another group of 24 chicks was kept as an uninoculated control group. The chicks were observed daily for clinical signs and mortality. Isolation of salmonella was done from different organs at 7 and 28 days postinoculation (DPI). All the chicks were weighed individually at 7, 14, 21, and 28 DPI. Two chicks chosen at random from each group were euthanatized and necropsied at 7 and 14 DPI and all the remaining live chickens, at 28 DPI. Selected tissues were taken for histopathology at 7 and 14 DPI. Dead chicks were examined for gross lesions and tissues were collected for histopathology. Chicks inoculated with S. pullorum had the highest mortality (66.66%), followed by S. typhimurium (33.33%). Chicks inoculated with S. heidelberg (00-1105-2) and SE PT4 (chicken-CA) had 12.5% mortality and 8.3% mortality, respectively, with SE PT 13a. Ceca were 100% positive for salmonellae at acute or chronic infection compared with other organs. Mean body weight reduction ranged from 0.67% (inoculated with S. kentucky 00-926-2) to 33.23% (inoculated with S. typhimurium 00-372) in the inoculated groups at different weeks compared with uninoculated controls. Gross and microscopic lesions included peritonitis, perihepatitis, yolk sac infection, typhilitis, pneumonia, and enteritis in some groups, especially those inoculated with S. typhimurium, S. heidelberg (00-1 105-2), SE PT4 (chicken-CA), and S. pullorum.     

Detection of chicken anemia virus DNA in the thymus of naturally infected chicks in turkey.

In this study, 94 clinically ill 11-28-day-old chicks belonging to eight broiler units from the Marmara region were investigated clinically for changes in hematocrit values and for the presence of chicken anemia virus (CAV) DNA. CAV DNA was detected by polymerase chain reaction in the thymus of 8.5% of the chicks. These chicks showed clinical signs of diarrhea, anorexia, depression, and growth retardation. The hematocrit values of these chicks were between 24% and 38%. At necropsy, hemorrhages were observed in the leg and pectoral muscles. Atrophy was noted in the thymus and in the bursa of Fabricius of positive chicks, and hemorrhages in the proventriculus of one positive chick were observed. This report describes the first detection of CAV DNA in chicks in Turkey.