Zinc bacitracin enhances colonization by the intestinal spirochaete Brachyspira pilosicoli in experimentally infected layer hens

Brachyspira pilosicoli strain CPSp1 isolated from a chicken in a broiler breeder flock in Queensland was used to experimentally infect 40 individually caged 22-week-old laying hens. Another 10 birds were sham-inoculated with sterile broth. All chickens received a commercial layer diet, but 10 infected birds had 50 parts/10 6 zinc bacitracin (ZnB) incorporated in their food. Birds were kept for 7 weeks, and faecal moisture, egg numbers, egg weights and body weights were recorded weekly. B. pilosicoli was isolated from the faeces of only three of the 30 inoculated birds receiving the diet without ZnB, whereas seven of the 10 inoculated birds receiving ZnB in their diet were colonized. This difference in colonization rate was highly significant ( P = < 0.001). Dietary ZnB at 50 parts/10 6 therefore predisposed to colonization by B. pilosicoli. Despite colonization, no significant production differences were found between the birds in the three groups.     

Influence of in-feed zinc bacitracin and tiamulin treatment on experimental avian intestinal spirochaetosis caused by Brachyspira intermedia

Thirty individually caged layer hens were inoculated with Brachyspira intermedia , and 20 control birds remained unchallenged. Birds received a diet containing 100 parts/10 6 zinc bacitracin (ZnB), and were monitored for 10 weeks. B. intermedia was recovered sporadically from five of the inoculated birds, and there were no significant effects on body weight, faecal water or egg production. ZnB was presumed to be indirectly inhibiting spirochaete growth, and when removed from the diet, 18 of the 30 inoculated birds rapidly became culture positive. After 4 weeks, 10 of the 30 infected birds were treated with tiamulin at 25 mg/kg for 5 days, and 10 were returned to the diet containing ZnB. Birds receiving tiamulin became spirochaete negative and maintained their egg production, but re-infection occurred. The other 20 infected birds had a significant drop in egg production, but those receiving ZnB showed a reduced colonization by B. intermedia after 3 weeks.     

Experimental infection of broiler breeder hens with the intestinal spirochaete Brachyspira ( Serpulina ) pilosicoli causes reduced egg production

The pathogenic potential of the anaerobic intestinal spirochaetes Brachyspira ( Serpulina ) pilosicoli and Brachyspira innocens was evaluated in adult chickens. Thirty 17-week-old Cobb broiler breeder hens were individually caged in three groups of 10 birds. Control birds (group A) were sham inoculated with sterile broth medium. Birds in the other two groups (groups B and C) were inoculated, respectively, with an isolate of B. innocens or of B. pilosicoli . Birds were monitored daily, and killed at 41 weeks of age. Infection had no consistent effect on body weight gain, but inoculation with B. pilosicoli resulted in a transient increase in faecal water content. B. innocens infection had no effect on egg production, but B. pilosicoli infection caused a delayed onset of laying, and a highly significant reduction in egg production over the first 11 weeks of lay. This study confirms that B. pilosicoli can cause serious egg production losses in adult chickens, while B. innocens is not obviously pathogenic.     

Antimicrobial susceptibility testing of Brachyspira intermedia and Brachyspira pilosicoli isolates from Australian chickens.

Susceptibilities of predominantly Australian isolates of the pathogenic intestinal spirochaetes Brachyspira intermedia (n = 25) and Brachyspira pilosicoli (n = 17) from chickens were tested in agar dilution against four concentrations each of the antimicrobials tiamulin, lincomycin, tylosin, metronidazole, tetracycline and ampicillin. Based on available minimum inhibitory concentration (MIC) breakpoint values for Brachyspira hyodysenteriae or other Gram-negative enteric veterinary pathogens, isolates of both species generally were susceptible to tiamulin, lincomycin, metronidazole and tetracycline. Although not classed as resistant, four isolates of B. intermedia had an elevated MIC range for tiamulin (1 to 4 mg/l), 11 isolates of B. intermedia and five of B. pilosicoli had an elevated MIC range for lincomycin (10 to 50 mg/l), one isolate of B. pilosicoli had an elevated MIC range for tetracycline (10 to 20 mg/l), and one isolate of B. intermedia and five of B. pilosicoli had an elevated MIC range for ampicillin (10 to 50 mg/l). A clear lack of susceptibility to tylosin (MIC > 4 mg/l) was seen in 11 isolates each of B. intermedia and B. pilosicoli, and to ampicillin (MIC > 32 mg/l) in two isolates of B. pilosicoli. These data suggest that some resistance to common antimicrobials exists among intestinal spirochetes obtained from laying hens and supports the need of MIC data for clinical isolates before any treatment is considered.     

Prevalence and disease association of intestinal spirochaetes in chickens in eastern Australia

Faecal samples (n = 1786) from chickens in broiler breeder (n = 28), layer (n = 22) or broiler (n = 19) flocks in the eastern states of Australia were cultured for intestinal spirochaetes. Overall, birds in 42.9% of broiler breeder and 68.2% of layer flocks were colonized with spirochaetes, but no birds in broiler flocks were infected. Colonization rates in infected flocks ranged from 10 to 100% of birds sampled. Faeces from colonized flocks were on average 14% wetter than those from non-colonized flocks. There was a highly significant association between colonization with spirochaetes and the occurrence of wet litter and/or reduced production. A subset of 57 spirochaete isolates from birds in 16 flocks were identified to the species level using a panel of polymerase chain reaction tests. Isolates from nine (56%) of these flocks were spirochaetes that are known to be pathogens of poultry: Serpulina pilosicoli was isolated from birds from five flocks, birds from two flocks were infected with Serpulina intermedia, and in two other flocks both species were identified. Isolates from the other seven flocks belonged to other Serpulina species, which are currently of unknown pathogenicity. This study indicates that infections with intestinal spirochaetes are a common but currently under-diagnosed cause of wet litter and/or reduced egg production in broiler breeder and layer flocks in Australia.     

Experimental infection of laying hens with Serpulina intermedia causes reduced egg production and increased faecal water content

Serpulina intermedia strain HB60, isolated from an Australian hen with diarrhoea, was used to infect 10 individually caged 14-week-old laying hens. Another 10 birds were sham inoculated with sterile broth. Birds were kept for 16 weeks, and faecal water content, egg production and body weights recorded. Strain HB60 was isolated from the faeces of nine of the infected birds at irregular intervals throughout the experiment, and from their caeca at slaughter. Infected birds tended to be lighter and their faeces, on average, were significantly wetter (by 2.85%; P < 0.002) than those of the controls. Significant reductions in mean number of eggs laid (1.4/week; P < 0.002) and mean egg weights (1.16 g; P < 0.05) were recorded in infected birds. Colonization did not induce any characteristic pathological changes. S. intermedia is potentially an economically significant cause of reduced egg production, and wet faeces in layer and broiler breeder flocks.     

Maduramicin and tiamulin compatibility in broiler chickens

A total of 480 1-day-old Hybro broiler chickens were divided into five treatment groups (A: unmedicated control, B: maduramicin, C: maduramicin + tiamulin, D: monensin + tiamulin and E: tiamulin) to study the effect on performance parameters, organ weights, blood haematology and biochemistry, and histopathology of liver and selected striated muscles, when maduramicin at 5 parts/10(6) and monensin at 100 parts/10(6) were included in feed in starter and grower periods, and tiamulin 9 in water at 270 parts/10(6) the recommended therapeutic level, from day 28 to 31. Performance parameters were significantly and negatively affected by monensin but not by maduramicin after treatment with tiamulin. Histopathological examination of striated muscles showed no incompatibility of maduramicin + tiamulin, while lesions were found in the monensin + tiamulin treated group. It is concluded that the use of tiamulin to a therapeutic level for 3 consecutive days is compatible with the simultaneous presence of maduramicin in the feed of broilers.     

Experimental infection of newly weaned pigs with human and porcine strains of Serpulina pilosicoli.

Cultures of Serpulina pilosicoli 95/1000, isolated from a pig with porcine intestinal spirochetosis (PIS), and S. pilosicoli WesB, isolated from an Aboriginal child with diarrhea, were used to infect 5-week-old newly weaned pigs. Four of 12 pigs infected with strain 95/1000 and 2 of 12 pigs infected with strain WesB became colonized and developed watery, mucoid diarrhea within 2 to 11 days postinfection. Affected pigs all had moderate subacute mucosal colitis, with gross and histological changes similar to those previously reported in both natural and experimentally induced cases of PIS. Silver-stained histological sections of the colon and cecum from affected pigs demonstrated spirochetes within dilated intestinal crypts, where they were associated with neutrophilic exocytosis and mucus secretion. Sections from one pig infected with strain 95/1000 showed large numbers of spirochetes attached by one end to the colonic epithelium, a feature consistent with PIS. This study confirms the role of S. pilosicoli in the etiology of PIS and provides evidence that S. pilosicoli strains of human origin have pathogenic potential in an animal model.     

Intestinal spirochaetes (Brachyspira spp.) colonizing flocks of layer and breeder chickens in Malaysia

Avian intestinal spirochaetosis causes problems including delayed onset of lay and wet litter in adult chickens, and results from colonization of the caecae/rectum with pathogenic intestinal spirochaetes (genus Brachyspira). Because avian intestinal spirochaetosis has not previously been studied in South East Asia, this investigation was undertaken in Malaysia. Faecal samples were collected from 25 farms and a questionnaire was administered. Brachyspira species were detected by polymerase chain reaction in 198 of 500 (39%) faecal samples from 20 (80%) farms, including 16 (94%) layer and four (50%) breeder farms. Pathogenic Brachyspira pilosicoli was identified in five (29%) layer and two (25%) breeder farms whilst pathogenic Brachyspira intermedia was detected in nine (53%) layer and one (12.5%) of the breeder farms. Twelve (80%) layer farms had egg production problems and 11 (92%) were positive for Brachyspira: three (25%) for B. pilosicoli and six (50%) for B. intermedia. Of three breeder farms with egg production problems, one was colonized with B. pilosicoli. Three of ten layer farms with wet litter were positive for B. pilosicoli and six for B. intermedia. Of four breeder farms with wet litter, one was colonized with B. pilosicoli and one with B. intermedia. No significant associations were found between colonization and reduced egg production or wet litter, perhaps because so many flocks were colonized. A significant association (P = 0.041) occurred between a high prevalence of colonization and faecal staining of eggs. There were significant positive associations between open-sided housing (P = 0.006), and flocks aged >40 weeks (P < 0.001) and colonization by pathogenic species.     

Antimicrobial resistance in Brachyspira pilosicoli with special reference to point mutations in the 23S rRNA gene associated with macrolide and lincosamide resistance

A point mutation in the 23S rRNA gene causes macrolide and lincosamide resistance in Brachyspira hyodysenteriae. The possible occurrence of a similar mutation in Brachyspira pilosicoli was studied and the MICs of six antimicrobial agents for Swedish field isolates of B. pilosicoli were determined. Of 10 isolates with high MICs of macrolide and lincosamide antibiotics, six had a mutation in nucleotide position 2058 or 2059 in the 23S rRNA gene as compared to the wild type of Escherichia coli, whereas none of 10 tylosin-susceptible isolates were mutated in this region. The mutations found in position 2058 were A --> T transversions, and in position 2059 either A --> G transitions or A --> C transversions. The MICs at which 90% of the B. pilosicoli field isolates were inhibited by tylosin, erythromycin, clindamycin, virginiamycin, tiamulin, and carbadox, were >256, >256, >4, 4, 2, and 0.125 microg/ml, respectively. In conclusion, point mutations in positions 2058 and 2059 of the 23S rRNA gene can cause macrolide and lincosamide resistance in B. pilosicoli. Macrolide resistance is widespread among Swedish field isolates of B. pilosicoli. Notably also a few isolates with elevated MICs of tiamulin were found.     

Vertical transmission and clinical signs in broiler breeders and broilers experiencing adenoviral gizzard erosion

The present report documents an outbreak of adenoviral gizzard erosion in 22 broiler flocks in Germany. The clinical picture was characterized by uneven growth of affected broilers that resulted in considerably lower than average weight at slaughtering. Fowl adenovirus serotype 1 (FAdV-1) was isolated from gizzard lesions and histological examinations demonstrated FAdV-1-positive intranuclear inclusion bodies in gizzard epithelial cells of affected broilers by in-situ hybridization. Birds from all affected flocks originated from one broiler breeder farm. During production of affected birds, broiler breeders were between 27 and 32 weeks old. Enzyme-linked immunosorbent assay and specific virus neutralization assay of sera from parent birds demonstrated an acute FAdV-1 infection within the first 5 weeks of the production cycle. Clinically, broiler breeders exhibited a moderate fall in the hatchability of their chicks, while egg production remained normal. No further clinical signs could be observed. Genetically identical FAdV-1 strains were isolated from gizzards of embryos at the lowest point of hatchability and from affected broiler flocks raised on independent farms. For the first time, direct detection of viable FAdV-1 from gizzards of embryos and progenies of one FAdV-1-seropositive broiler breeder farm in the course of an outbreak of adenoviral gizzard erosion could be demonstrated, highlighting the importance of vertical transmission of this disease. Additionally, growth retardation and subsequent reduced average weight at the time of slaughter of broiler chickens underline the economic impact of adenoviral gizzard erosion for poultry production.     

Antibiotic sensitivity and resistance in Ornithobacterium rhinotracheale strains from Belgian broiler chickens

Establishing the antibiotic sensitivity of the avian respiratory pathogen Ornithobacterium rhinotracheale is difficult because of the organism's complex growth requirements and the unusually frequent occurrence of resistance. The minimal inhibitory concentrations of 10 antibiotics were determined for 45 strains of O. rhinotracheale from Belgian broiler chickens collected from 45 farms between 1995 and 1998. They were compared with the type strain, which was isolated from a turkey, and a strain isolated from a rook. All the broiler strains were resistant to lincomycin and to the β -lactams ampicillin and ceftiofur. Less than 10% of the strains were sensitive to the macrolides tylosin and spiramycin, tilmicosin and flumequine. A few strains were sensitive to enrofloxacin and doxycycline. All strains were sensitive to tiamulin.     

Treatment of a field case of avian intestinal spirochaetosis caused by Brachyspira pilosicoli with tiamulin.

There has been much confusion over the significance of spirochaetes found in the caeca of laying hens and the impact they may have on egg production. In recent years, the situation has been made clearer and the presence of such species as Brachyspira pilosicoli have been shown to cause a mild, chronic disease in both layers and breeders and to reduce egg production by reportedly 5%. In the United Kingdom, a multi-age caged laying site with three separate flocks of approximately 12 000 birds each was chronically infected with B. pilosicoli but displayed few clinical signs except for a noticeable reduction in egg production and an increased mortality. The flocks were treated for 3 days in the drinking water with tiamulin at 12.5 mg/kg bodyweight, and a steady improvement in performance was recorded. The production results were compared with a flock that had been untreated with tiamulin previously, as a control, and one that had been treated at 25 and 45 weeks of age. A 9.8% improvement in egg production/hen housed up to 72 weeks of age and 9.7% in total egg weight was recorded, as well as an 8.6% reduction in actual hen mortality, in the tiamulin-treated flock in comparison with the untreated control. After taking into account the difference in breeds used, there was only a 6% reduction in egg production but an 8.84% increase in mortality in the untreated flock compared with the individual breed's standard production data. The cost of the disease was estimated at 14 million pound in the United Kingdom, based on a national laying flock of 30 million or 1.5% of production. Faecal examination for potentially pathogenic spirochaetes should be part of the differential diagnosis of under-performing laying flocks.     

Effects of subfascial pressure in experimental deep pectoral myopathy of the fowl: an angiographic study

1. Angiography of the supracoracoid muscles of broiler breeder hens after neurostimulation of the breast muscles showed that complete ischaemia developed within 10 min and persisted for at least 1 h. After 24 h ischaemia remained only in the middle 1/4-1/3 of the muscle, coinciding with the main site of necrotic lesions found in experimental and spontaneous deep pectoral myopathy. 2. The development of ischaemia was prevented when the deep pectoral fascia was incised along its length before neurostimulation. 3. An increase in subfascial pressure in the supracoracoid was observed during 30 min after neurostimulation in both broiler breeder and lightweight hens. This was greater in the broiler breeder hens and more than sufficient to arrest blood flow. 4. During the first hour after neurostimulation an increase of 20% occurs in the weight of the supracoracoid muscle of both types of hen but only the broiler hens developed ischaemia. 5. It is suggested that the immediate cause of the ischaemia following neurostimulation is a rise in intramuscular pressure due to the increased muscle volume within an osteofascial compartment of limited size. The basic difference between the two types of bird in their susceptibility to this type of ischaemia and to deep pectoral myopathy probably lies in the relative development of the supracoracoid and its overlying fascia.     

Anaemia-dermatitis of broilers: Field observations on its occurrence, transmission and prevention

Anaemia-dermatitis was first observed in German broiler flocks in 1977. Its frequency has increased in the past six years. Atrophy of thymus, bursa and bone marrow occur and are affected by a severe anaemia and immunosuppression. Secondary bacterial infections of the skin cause gangrenous dermatitis. Systematic investigations of outbreaks in two broiler integrations showed the syndrome to occur only in the offspring of young broiler breeders during the first 3 to 9 weeks of production. Anaemia could be reproduced experimentally in CAA-antibody negative SPF birds by injecting a bacteria-free filtrate of organ homogenates of diseased birds; birds kept in contact with the inoculated chicks remained healthy. It is concluded that anaemia-dermatitis is primarily caused by the chicken anaemia agent (CAA). Vertical transmission via hatching egg predominates with no evidence of horizontal transmission. In order to prevent egg transmission of CAA immunisation during rearing is indicated for breeder stocks.     

Further studies on vertical transmission and persistence of Salmonella enterica serovar Enteritidis phage type 4 in chickens

One-week-old commercial layers were infected orally with 10⁸ colony forming units of Salmonella enterica serovar Enteritidis phage type 4. No mortality was observed. The inoculated organism was isolated in decreasing viable numbers from a number of tissues, particularly the spleen, liver and caeca. Organisms present in the spleen were primarily localized within macrophages. No Salmonella Enteritidis organisms were isolated between 10 and 24 weeks of age, when the experiment was terminated after several weeks of lay. When two groups of adult hens, housed with males, were infected, contaminated eggs were found within 2 weeks of infection in one of the experiments only. Progeny hatched from these eggs showed no mortality unless they were infected artificially with the S. Enteritidis strain. In this case, the percentage mortality fell as the hatches progressed, indicating increasing immunity to infection. The faecal excretion of the inoculated phage type 4 strain by infected but healthy progeny was followed. Although most birds ceased to excrete by 11 to 12 weeks of age, a small number of the birds continued to excrete until they themselves came into lay. The small numbers of birds in which this occurred indicates that tolerance to infection does not occur readily following infection of hens laying fertile eggs or in progeny birds infected before or within hours of hatching. Birds infected when they were less than 24 h old remained persistently infected until they were well into lay. However, control birds infected when 1 week old, on this occasion, showed a high level of excretion until the birds began to lay at 18 weeks. Inbred lines of chickens showing differences in their susceptibility to systemic salmonellosis did not show significant differences in the extent to which S. Enteritidis localized in the organs of the reproductive tract or in the number of infected eggs produced.     

Egg transmission of a respiratory isolate of Mycoplasma synoviae and infection of the chicken embryo

Egg transmission of Mycoplasma synoviae (Ms) was demonstrated in broiler breeder birds of over 40 weeks of age from a commercial flock that had been infected during rearing and in birds experimentally inoculated before onset of lay. Infection of growing birds with Ms did not prevent egg transmission in adult life. The embryonated eggs of experimentally infected hens were less susceptible to infection by inoculation than those of specific pathogen-free birds. Some chicks with maternal antibody were found to be infected at hatching and in these maternal antibody was not mycoplasmacidal. Indeed by protecting the embryo maternal antibody may have promoted hatching of infected chicks.     

Induction of eggshell apex abnormalities in broiler breeder hens

Recently, the causal relationship between eggshell apex abnormalities (EAA) and Mycoplasma synoviae was described. This eggshell pathology has only been documented in table egg layers both spontaneously and experimentally infected with M. synoviae, suggesting that meat-type layers are less prone to this condition. In this study the susceptibility of specified pathogen free (SPF) broiler breeder hens to produce eggs with EAA after M. synoviae infection was assessed. Five groups of 12 hens each were made: a negative control group, a group inoculated intratracheally (i.t.) with a M. synoviae EAA strain at 19 weeks of age, a group inoculated i.t. with this strain at 19 and 26 weeks of age, a group inoculated with M. synoviae i.t. at 19 weeks of age and infected 5 days earlier with infectious bronchitis virus D1466 (IBV), and a fifth group similar to the former but inoculated i.t. twice with an M. synoviae EAA strain at 19 and 26 weeks of age. Eggs with EAA were only produced after a single i.t. inoculation with the M. synoviae EAA strain if preceded by an infection with IBV. The production of eggs with EAA started 6 weeks after M. synoviae EAA inoculation and the proportion of eggs with EAA during the experiment was 9/449 (2%), which was much lower than that in SPF layer hens (14–22%). The present results suggest that broiler breeder hens are less susceptible to producing eggs with EAA after an infection with a M. synoviae EAA strain preceded by an IBV infection, compared with table egg layers. Similar to SPF egg layers, the mean daily egg production per hen was significantly reduced by the M. synoviae EAA strain and there was a general negative effect on eggshell strength by this strain, suggesting it could also have a detrimental effect on hatching egg quality.     

The detection of the big liver and spleen agent in infected tissues via intravenous chick embryo inoculation

Partially purified homogenates prepared from the livers of birds affected with BLS were used to inoculate intravenously young adult broiler breeder hens and incubating chicken embryos. Following inoculation, the broiler breeders developed pathological and serological responses typical of BLS infection. The same antigen resulted in persistent antigenaemia in chickens which were inoculated during various stages of incubation. This technique was very sensitive and could be used for detection and titration of virus in tissue preparations. Methods for optimization of the technique were investigated. Chick embryos demonstrated the greatest susceptibility to agent replication when inoculated at 11 days of embryonation. After hatching, agent replication appears to increase with age. Inoculated chickens bled at 8 days of age showed increased incidence of antigenaemia by this procedure.     

Mycoplasma iowae infection in broiler breeders

Experimental infection of broiler breeder chickens at one day of age with two strains of Mycoplasma iowae resulted in stunting and poor feathering accompanied, in some cases, by rupture of the digital flexor tendon, tenosynovitis, tendon fibrosis and arthritis. Broiler breeder birds appeared to be more susceptible to the effects of infection than were light, layer-type birds inoculated with the same two strains in an earlier experiment. The two strains of organism did not persist in the affected birds nor did they consistently stimulate the production of agglutinating antibodies.