Haematological, serum biochemical and serological features of platypuses with and without mycotic granulomatous dermatitis

OBJECTIVE: To determine whether there are haematological, serum biochemical and serological differences between platypuses (Ornithorhynchus anatinus) with and without granulomatous dermatitis due to Mucor amphibiorum infection. An additional objective was to establish reference haematological and serum biochemical ranges for the species in Tasmania. DESIGN: A clinicopathological and serological study. ANIMALS: A total of 37 free-living adult platypuses captured from streams and dams in Northern Tasmania were used in the clinicopathological study. Twenty-seven were clinically normal and 10 had mycotic granulomatous dermatitis. A total of 22 platypuses (20 adult and 2 juvenile) were used for the serosurvey. Eighteen were captured from streams in Northern Tasmania, and four were submitted for necropsy. RESULTS: Platypuses with mycotic ulcerative dermatitis had significantly smaller packed red cell volumes, haemoglobin concentrations, lymphocyte counts, serum cholesterol and calcium concentrations, and higher serum globulin and potassium concentrations than clinically normal animals. The lymphopenia and hyperkalaemia were thought to be clinically significant. Numbers of Trypanosoma binneyi in blood smears were similar between the two groups. Diseased platypuses had higher concentrations of serum antibody against Mucor amphibiorum as determined by ELISA compared to clinically normal platypuses. CONCLUSION: Platypuses affected by mycotic granulomatous dermatitis showed haematological and serum biochemical changes when compared to clinically normal animals from the same Tasmanian sites. A serological survey may be a useful method for detecting the prevalence of exposure to Mucor amphibiorum and humoral immunity in platypus populations both in Tasmania and the mainland of Australia.     

Frequency of yeasts and dermatophytes from healthy and diseased dogs.

The aim of this study was to investigate the presence of dermatophytes and yeasts in healthy and diseased dogs. A total of 633 samples were collected from 26 healthy animals (104 samples), 131 with dermatitis (343 samples), 74 with otitis (148 samples), and 19 with ocular diseases (38 samples). Cultures from healthy animals were positive for Malassezia pachydermatis in 13.5% (7/52) of samples from skin, 42.3% (11/26) from ear, and 3.8% (1/26) from eye. Fungal growth was observed in 20.4% (70/343) samples from animals with dermatitis. Microsporum canis was the most isolated fungus (n = 39), followed by M. pachydermatis (n = 30) and Malassezia sp. (n = 3). Of the 148 samples from dogs with otitis, 90 (60.8%) were positive for M. pachydermatis, and of the clinical specimens from the conjunctiva of animals with ophthalmic disease, 2.6% (1/38) presented positive cultures for M. pachydermatis. Only 14.3% (2/14) of the positive cultures for M. pachydermatis and 40.9% (9/22) of those for M. canis were positive in the direct exam. Direct exams were positive in 84.3% (70/83) of the culture positive samples from affected ears of dogs with otitis. Malassezia pachydermatis may act as an aggravating factor in the occurrence of cutaneous diseases, or the isolation of M. canis may be associated with the onset of dermatophytosis. Fungal culture, rather than microscopic examination, should be used as the definitive diagnostic test for dermatomycoses and otitis.     

Preliminary study using an indirect ELISA for the detection of serum antibodies to Alternaria in domestic cats

Alternaria is a saprophytic fungus that is widespread in the environment; it is an opportunistic pathogen and causes disease in human beings and domestic animals. Fungal spores gain entry to the host through skin lesions and cause slow-growing, soft to firm, subcutaneous swellings, either with or without ulcers. An indirect ELISA was developed for the detection of anti-Alternaria immunoglobulin G (IgG) antibodies in serum to determine the prevalence of Alternaria exposure in domestic cats. Fifty-two of 63 cats had detectable levels of anti-Alternaria IgG antibody. There were no correlations between the concentration of antibody and the sex, breed or living environment of the cats, but cats less than two years of age had significantly lower concentrations than older cats. The cats with disease caused by culture-confirmed Alternaria infections did not have significantly higher concentrations of antibody than the healthy cats or cats with other diseases.     

Clostridium perfringens alpha-toxin and NetB toxin antibodies and their possible role in protection against necrotic enteritis and gangrenous dermatitis in broiler chickens

Necrotic enteritis (NE) and gangrenous dermatitis (GD) are important infectious diseases of poultry. Although NE and GD share a common pathogen, Clostridium perfringens, they differ in other important aspects such as clinical signs, pathologic symptoms, and age of onset. The primary virulence factors of C perfringens are its four major toxins (alpha, beta, epsilon, iota) and the newly described NE B-like (NetB) toxin. While neutralizing antibodies against some C perfingens toxins are associated with protection against infection in mammals, the serologic responses of NE- and GD-afflicted birds to these toxins have not been evaluated. Therefore, we measured serum antibody levels to C perfringens alpha-toxin and NetB toxin in commercial birds from field outbreaks of NE and GD using recombinant toxin-based enzyme-linked immunosorbent assay (ELISA). Initially, we used this ELISA system to detect antibody titers against C perfringens alpha-toxin and NetB toxin that were increased in birds experimentally coinfected with Eimeria maxima and C perfringens compared with uninfected controls. Next, we applied this ELISA to field serum samples from flock-mated birds with or without clinical signs of NE or GD. The results showed that the levels of antibodies against both toxins were significantly higher in apparently healthy chickens compared to birds with clinical signs of NE or GD, suggesting that these antitoxin antibodies may play a role in protection against NE and GD.     

Detection of spirochetes by polymerase chain reaction and its relation to the course of digital dermatitis after local antibiotic treatment in dairy cattle

The aim of the study was to monitor the course of digital dermatitis after local antibiotic treatment in an experimental group (treated on diagnosis) and a control group (treated 5 days later). The present study was carried out on 2 farms involving 18 animals. Monitoring was performed by means of clinical findings and detection of spirochetes on the surface of the lesions, using a polymerase chain reaction. Superficial wound smears were taken before and after treatment. Twelve animals on both farms followed the classical healing process, but six animals responded poorly to treatment. We observed that without treatment, there was no self-cure in the control group within 5 days. There was a significant improvement in the clinical condition of all animals after treatment on both farms, during the follow-up period. The time until reappearance of new digital dermatitis lesions was not significantly different between the experimental and control group, but it was different between the two farms which could be due to the influence of farm factors. Using primers specific for Treponema denticola and Treponema vincentii, all the disease stages had at least one positive polymerase chain reaction result indicating the presence of spirochetes in samples of all the disease stages during the healing process. This implies that the spirochetes are not completely eradicated from the surface of the lesions after treatment. It was also observed that the classical ulcerative disease stage (M2) had relatively more positive polymerase chain reaction results compared to any other disease stage, showing a possible link between the presence of spirochetes and clinical disease.     

Infectious bovine keratoconjunctivitis: Bacteriologic,immunologic, and clinical responses of cattle to experimental exposure with Moraxella bovis

The bacteriologic, immunologic, and clinical responses of 3- to 4-month old Holstein-Friesian calves to experimental exposure with Moraxella bovis type 10900 has been investigated. After u.v. radiation and intraconjunctival exposure with 1.9 × 10⁷ microorganisms, each eye of 16 calves exhibited signs of blepharospasm, photophobia, and increased lacrimation. Bacteria were recovered from exposed eyes for 2–7 consecutive weeks before maximal clinical response occurred. The severity of the cases varied from eyes that exhibited mild signs to severe clinical cases with profuse lacrimation, conjunctival swelling, corneal opacity, and ulceration. By 70 days after exposure, M. bovis could not be recovered from any conjunctival swabs, and clinical signs were not observed. Four non-exposed control animals did not develop clinical signs nor was M. bovis recovered from conjunctival swabs.Lacrimal secretions collected at the time of and 1 week after maximal clinical response had significantly elevated levels of total protein as compared to those collected 3, 2, and 1 week before, and 2 and 3 weeks after maximal clinical response. A passive hemagglutination test, using tanned formalized sheep erythrocytes sensitized with M. bovis sonicate antigen, detected antibody in lacrimal secretions from 22 of 32 eyes. The appearance of specific antibody in lacrimal secretions correlated with the amelioration of clinical signs and the decline in numbers of M. bovis microorganisms recovered from conjunctival swabs.     

A longitudinal study of bovine coronavirus enteric and respiratory infections in dairy calves in two herds in Ohio

This prospective longitudinal study examined the epidemiology and disease syndrome associated with bovine coronavirus (BCV) infections in a cohort of 8 conventional calves from 0 to 120 days of age, in two dairy herds in Ohio. The periods of respiratory shedding of BCV were determined by direct immunofluorescent (DIF) staining of nasal epithelial cells and ELISA of nasal swab supernatant fluids. The periods of fecal shedding of BCV were determined by ELISA and immunoelectron microscopy (IEM). The isotype-specific antibody titers to BCV in serum (at selected intervals between 0 and 120 days of age) and the post-suckling (24 to 48 h after birth) total immunoglobulin levels were examined by ELISA and zinc sulfate turbidity tests, respectively. Of the 8 calves studied, 4 had evidence of BCV respiratory (by DIF or ELISA) or enteric infections (by IEM or ELISA) in association with diarrhea or rhinitis, even though 7 of 8 calves showed increases in one or more serum antibody isotypes to BCV and 6 of 8 calves showed BCV respiratory or enteric antigen shedding by ELISA. Serological antibody titer increases occurred in 3 calves before 30 days of age and in 4 calves after 30 days of age; two of the latter calves had a second rise in serum antibody titers to BCV after the initial rise. A serological antibody titer increase was not observed in one calf. This suggests that BCV infections may be very common in a closed herd and may occur in older calves, although many may be subclinical and some may be recurrent. There were no statistically significant correlations between total serum immunoglobulin levels or BCV antibody isotype titers in serum (24-48 h after birth) and clinical disease or infection by BCV; however, calves with low levels of IgA BCV antibodies in serum (24-48 h after birth) had a significantly greater average number of days with diarrhea than those calves having high levels of IgA BCV-specific antibodies in serum.     

Serum immunoglobulin E concentrations in West Highland White Terrier puppies do not predict development of atopic dermatitis

Sera from 154 West Highland White Terrier puppies between 6 and 12 weeks of age were assayed for total IgE using a sandwich ELISA method. Development of clinical signs of atopic dermatitis in these dogs was monitored by use of an annual owner questionnaire, until the dog reached 3 years of age. Of 114 evaluated dogs, skin disease severe enough to warrant veterinary examination was reported in 52 (46%) during the three study years. A diagnosis of atopic dermatitis was made by the attending veterinarian in 28 dogs (25%). Certain litters had an especially high prevalence of apparently atopic dogs, consistent with the genetic predisposition towards atopy in this breed, but clear evidence of consistent heritability was not present. The median IgE concentration in 154 puppies at 6–12 weeks of age was 0.9 units mL⁻¹, with a skewed distribution. Significant ( P < 0.01) variation in serum IgE concentrations was observed between litters, with median serum IgE concentrations for a litter ranging from 0 to 27.7 units mL⁻¹. The median serum IgE concentration in puppies that later developed clinical signs of atopic dermatitis was not significantly different from that of puppies that remained healthy. There were no apparent correlations or significant differences found between serum IgE concentration as a puppy, parental history of skin disease, and subsequent emergence of clinical signs of atopic dermatitis. We conclude that early total serum IgE determinations seem to have little usefulness in predicting the later onset of atopic dermatitis in this breed.     

Development of canine systemic lupus erythematosus model

The purpose of this study was to develop a model for canine systemic lupus erythematosus. Systemic lupus erythematosus (SLE) is a systemic autoimmune syndrome defined by clinical and serological features, including arthritis, glomerulonephritis, dermatitis and autoantibodies. SLE was induced in eight normal dogs by immunization with heparan sulphate, the major glycosaminoglycan of the glomerular basement membrane. All the heparan sulphate-immunized dogs showed mild-to-moderate levels of proteinuria and skin disease. Cutaneous signs associated with SLE including alopecia, erythema, crusting, scaling and seborrhoea were observed. Immunohistological examination of the skin lesions revealed deposition of immunoglobulin M and complement in the dermal-epidermal junction. Three of eight dogs showed lameness. The antinuclear antibody tests were positive with the antibody titres higher than 1:128. Therefore, this experimental SLE model could be useful for studying immune-mediated skin disease and autoimmunity.     

Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens

Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with immune sera from commercial meat-type chickens with clinical outbreak of Clostridium infections. In addition to the genes encoding EF-Tu and PFO, C. perfringens alpha-toxin and necrotic enteritis B-like (NetB) toxin were also expressed in Escherichia coli and their corresponding recombinant proteins were purified. Using the four recombinant proteins as target antigens in ELISA immunoassays, high serum antibody titers were observed not only in chickens with clinical signs of Clostridium infections, but also in apparently healthy animals from the same disease-endemic farm. By contrast, no antibodies against any of the proteins were present in the serum of a specific pathogen-free bird. In ELISA using recombinant proteins of C. perfringens, the levels of anti-bacterial protein antibodies were also higher in chickens which were experimentally induced to show NE clinical signs after co-infection with C. perfringens and Eimeria maxima compared with uninfected controls. These results show that two antigenic C. perfringens proteins, EF-Tu and PFO can be useful detection antigens for C. perfringens-afflicted infections in commercial poultry.     

The application of the ELISA to the diagnosis and control of avian encephalomyelitis

An ELISA for measuring serum antibody against avian encephalomyelitis virus (AEV) was evaluated for its application to the diagnosis and control of avian encephalomyelitis (AE). A scoring system was developed for this ELISA (AE ELISA-Index) so that the overall level of antibody in the flock could be presented in a single, convenient number. During suspected outbreaks of disease thought to have been caused by AEV infection, the AE ELISA-Index increased in sequential serum samples. High levels of antibody against AEV were measured in 13 flocks experiencing egg productivity problems. Variable levels of antibody activity against infectious bronchitis virus (IBV) were also observed in 11 of these flocks. The AE ELISA-Index was correlated with the embryo susceptibility test. Application of the AE ELISA has indicated that natural exposure to the virus does not occur in all flocks, and vaccination failures were detected sufficiently early for revaccination to be administered before the onset of lay.     

Preliminary investigation into the prevalence of mucormycosis in the platypus (Ornithorhynchus anatinus) in three catchments in north-west Tasmania

Objective To investigate the distribution and prevalence of mucormycosis in platypus (Ornithorhynchus anatinus) from the Inglis, Emu and Black-Detention catchment areas in north-west Tasmania. Procedure A field study was performed and resulted in the examination of 44 wild platypuses; in addition, one dead platypus and two live platypuses were examined after they were independently submitted to a local veterinary clinic. Results No cases of mucormycosis were conclusively diagnosed. One platypus with signs consistent with those previously described in cases of mucormycosis was captured in the Emu River catchment. However, laboratory tests did not provide a definitive diagnosis for the lesion. Two platypuses from the Inglis catchment area had signs very similar to those previously described in cases of mucormycosis, but laboratory tests found Corynebacterium ulcerans to be the likely cause of the cutaneous ulcers on one of these platypuses and an unidentified fungal agent to be the cause of a cutaneous nodule in the other. Conclusions These findings do not prove that mucormycosis is absent from the populations studied. However, they may indicate that the prevalence of disease is low. The possibility that Mucor amphibiorum is present in a subclinical form in platypuses, or infecting another reservoir, is not excluded. The findings also suggest that caution should be exercised when diagnosing mucormycosis based on clinical findings alone and raise the possibility that some cases may have been incorrectly diagnosed.     

Quantitation of canine distemper virus and antibodies by enzyme-linked immunosorbent assays using protein A and monoclonal antibody capture

Monoclonal antibodies produced from 19 cloned hybridomas were selected for this study. Specific canine distemper virus (CDV) antibodies in medium from cloned hybridomas were detected by direct enzyme-linked immunosorbent assays (ELISA) and by indirect immunofluorescence. Three different sandwich ELISA systems were developed either to detect CDV in cell cultures and clinical specimens or to detect specific antibody in canine sera. Protein A and monoclonal antibodies attached in sequence to a solid phase constituted the capture system in the assays. Viral antigens were detected by sandwiching extracts of clinical specimens (or infected cell cultures), monoclonal antibody, and peroxidase-labeled protein A in sequence onto the capture layer. In 1 procedure, biotin-labeled antibody and peroxidase-labeled avidin were used as the last 2 layers in the assay. The CDV antibodies in dog sera were quantitated in a similar manner, but the sequential sandwiching levels consisted of partially purified CDV, serum specimen, and peroxidase-labeled protein A, respectively. The procedures were specific and highly sensitive.     

Subclinical infectious bursal disease in an integrated broiler production operation.

A field study was designed to determine the prevalence of subclinical infectious bursal disease (IBD) in broiler chickens from a commercial poultry company. Bursae of Fabricius (BF) from two vaccinated and three nonvaccinated broiler flocks were evaluated histologically, and antibody profiles of these broiler and matched parent breeder flocks were established. Lesions of IBD, including lymphoid necrosis, stromal edema, and infiltrates of heterophils and macrophages, were first detected in BF at 24 days of age in both vaccinated and nonvaccinated chickens. At 41 days, all BF had lesions characteristic of IBD, including severe lymphoid depletion, proliferation of epithelial cells, and mild fibroplasia. Although mean maternal antibody levels (measured by enzyme-linked immunosorbent assay) in broilers were apparently protective through day 12, IBD antibodies decreased to nonprotective levels (below 1,000) by day 16 or 20. Titers began to increase by day 28 or 32 because of field exposure. Sentinel birds, placed with broiler flocks, also developed IBD antibody titers. Broiler breeders had low and nonuniform antibody titers. Prevalence of field IBD exposure was high, and existing vaccination programs were not effective.     

Ulcerative and papillomatous digital dermatitis of the pastern region in dairy cattle: clinical and histopathological studies.

The study was carried out on a single herd of 500 Holstein breed dairy cattle. Only 50 dairy cows (10%) were found to be suffering from focal ulcerative and papillomatous digital dermatitis of the pastern region. The cows were subjected to clinical and histopathological examinations. Early ulcerative stage was seen in 30 cows and late papillomatous stage was observed in 20 cows. The topical application of oxytetracycline solution or benzathine penicillin powder appeared to be effective for the treatment of the ulcerative lesions. The histopathological findings showed signs of epidermopoiesis and papilloma formation. The dermal reaction revealed signs of fibroplasia and perivascular aggregation of inflammatory cells. Silver stained sections indicated the presence of longer filamentous spiral spirochetes, cocci and large-sized anaerobic bacilli invading the stratum spinosum. In conclusion it can be said that bovine digital dermatitis was observed in ulcerative and papillomatous form at the pastern region bordering the dewclaws. Spirochetes are frequently associated with and may be responsible for pathological changes in the lesion.     

The survival of platypuses in captivity

Data are presented on the duration of survival of 228 platypuses at six Australian zoos between 1934 and 1988. Only 22.4% of all platypuses survived more than 1 year in captivity. Of 15 living platypuses, 3 had been held in captivity for less than 1 year, 5 for between 1 and 5 years, 6 for between 5 and 10 years and 1 for 21 years. Of 213 platypuses that died in captivity, 81.7% had died within 1 year; most within the first month. The duration of survival was unrelated to the age of animals at acquisition or to sex. The survival rate of animals donated to zoos, including "refugees", was similar to that of purpose-caught animals. Clearly, only a small proportion of platypuses adapted to captive husbandry. The cause of death of most platypuses was not established. However, infectious disease did not appear to be significant. Approximately 28% of deaths were related to inadequate husbandry. Recommendations are made to improve the survival of platypuses in captivity. Research has commenced in zoos to facilitate this goal.     

Sequential bacteriological observations in relation to cell-mediated and humoral antibody responses of cattle infected with Mycobacterium paratuberculosis and maintained on normal or high iron intake

Twenty calves were orally infected with Mycobacterium paratuberculosis before weaning. Ten of these plus 4 non-infected controls were maintained on elevated dietary iron intake from 6 to 33 months of age. During this time, in which the majority of animals were bred, the influence of increased dietary iron upon tests of cellular and humoral immune responsiveness to antigens of the organism were monitored. Results were examined in relation to the organism's capacity to multiply and infect up to 7 portions of the intestinal tract. No significant differences were detected in the degree of intestinal disease or pattern of faecal excretion of M. paratuberculosis in iron supplemented and non-supplemented cattle. Cutaneous delayed-type hypersensitivity (DTH) to johnin PPD developed at 1 month and in-vitro lymphocyte and immunostimulatory activity (LS) to this antigen at 2 months after infection. LS indices were significantly reduced in magnitude in iron-supplemented cattle (p less than 0.01). Most ELISA antibody responses were positive 10 to 17 months after infection and preceded the fewer number of CF responses by several months. Neither of the antibody tests was affected by elevated iron intake. Generally, complete or partial resistance to paratuberculosis was associated with sustained positive monthly LS tests (index greater than or equal to 2.0), whereas antibody levels tended to be sustained only in the more severely affected cattle. Although neither test system was affected by pregnancy the ELISA failed to detect a significant proportion of cattle chronically shedding M. paratuberculosis in faeces.     

Application of different methods for the diagnosis of paratuberculosis in a dairy cattle herd in Argentina

Paratuberculosis (Ptbc) has a high prevalence in Argentina, that affects dairy and beef cattle. The culture is the gold standard to the diagnosis of the disease. Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis), the aetiological agent, is difficult to isolate and grow in culture. In this study, 24 randomly selected cows of the Fresian breed from a dairy herd with a history of Ptbc were used to evaluate the performance of different diagnostic techniques. These animals did not show clinical signs of the disease. However, another animal from this herd presented evidence of clinical disease at the moment of the present study. This animal was necropsied and one strain of M. paratuberculosis was isolated from faeces, lymph nodes and intestine. Serum for indirect absorbed enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) tests and whole blood samples to perform gamma interferon (gammaIFN) release assays were obtained from each animal. Faeces and milk samples to carry out bacteriological cultures, PCR identification of M. paratuberculosis, and direct examinations of smears with Ziehl-Neelsen's (ZN) stain were also collected. Tuberculin test with bovine purified protein derivative (PPD) in the caudal fold was performed. The results showed that 10 out of 24 animals (41.6%) were positive to ELISA. Eight strains of M. paratuberculosis were isolated, six from faeces, two from milk. Five of the animals that excreted the bacteria through faeces were ELISA-positive, whereas the excreters through milk were negative to ELISA. No positive samples by AGID were obtained in clinical asymptomatic animals. Seven samples gave positive gammaIFN results with avian PPD, but only two of these animals were confirmed with culture. Direct PCR, to detect IS900 (M. paratuberculosis) in faeces and milk samples, was negative, but PCR using material taken from faecal and milk cultures gave positive results before visualizing the colonies. No sample was positive by PCR directed to IS6110 (M. tuberculosis complex). There was not always agreement between isolations and ZN in the studied samples. In conclusion, the absorbed ELISA was useful to detect positive animals and excreters through faeces but not through milk. PCR applied to cultures with incipient development before the visualization of colonies was effective to specifically determine the presence of M. paratuberculosis. The gammaIFN test was not able to detect the most positive animals confirmed by culture. The importance of using ELISA and cultures is emphasized by this study but it is necessary to continue with the gammaIFN test development for early detection of the disease.