Antibiotic resistance in Campylobacter jejuni and Campylobacter coli isolated from poultry in the South-East Queensland region

Objectives: The aim of this study was to determine the antimicrobial resistance patterns of 125 Campylobacter jejuni and 27 Campylobacter coli isolates from 39 Queensland broiler farms. Methods: Two methods, a disc diffusion assay and an agar-based MIC assay, were used. The disc diffusion was performed and interpreted as previously described (Huysmans MB, Turnidge JD. Disc susceptibility testing for thermophilic campylobacters. Pathology 1997; 29: 209–16), whereas the MIC assay was performed according to CLSI (formerly NCCLS) methods and interpreted using DANMAP criteria. Results: In both assays, no C. jejuni or C. coli isolates were resistant to ciprofloxacin or chloramphenicol, no C. coli were resistant to nalidixic acid, and no C. jejuni were resistant to erythromycin. In the MIC assay, no C. jejuni isolate was resistant to nalidixic acid, whereas three isolates (2.4%) were resistant in the disc assay. The highest levels of resistance of the C. jejuni isolates were recorded for tetracycline (19.2% by MIC and 18.4% by disc) and ampicillin (19.2% by MIC and 17.6% by disc). The C. coli isolates gave very similar results (tetracycline resistance 14.8% by both MIC and disc; ampicillin resistance 7.4% by MIC and 14.8% by disc). Conclusions: This work has shown that the majority of C. jejuni and C. coli isolates were susceptible to the six antibiotics tested by both disc diffusion and MIC methods. Disc diffusion represents a suitable alternative methodology to agar-based MIC methods for poultry Campylobacter isolates.

The vaccination-challenge trial: the gold standard test to evaluate the protective efficacy of infectious coryza vaccines

Infectious coryza is an upper respiratory tract disease of chickens with the major impact occurring in multi-age flocks. We investigated the relationship between the level of antibodies, as detected by a haemagglutination-inhibition (HI) assay, in infectious coryza-vaccinated chickens and the protection against challenge in those chickens. In one experiment, chickens given a single dose of either of two infectious coryza vaccines lacked a detectable HI response to vaccination but showed significant levels of protection 11 weeks after vaccination. In contrast, in chickens given two doses of an infectious coryza vaccine and challenged 3 weeks after the second vaccine dose, there was a strong serological response with 36/40 birds having a HI titre of 1/20 or greater. In this trial there was an apparent relationship between titre and subsequent protection, with none of the 32 chickens with a titre of 1/40 or 1/80 showing any clinical signs and only one of the same group yielding the challenge organism on culture. In contrast, three of the four vaccinated chickens with a HI titre less than 1/5 developed the typical clinical signs of coryza and yielded the challenge organism on culture. Overall, our results suggest that HI titres cannot be regarded as a definitive predictor of vaccine efficacy. We suggest that the vaccination-challenge trial is the gold standard for the evaluation of the immune response to infectious coryza vaccines.

Detection of Chlamydiaceae DNA in veterinary specimens using a family-specific PCR

Aims: The aim of this work was to develop a rapid molecular test for the detection of the Chlamydiaceae family, irrespective of the species or animal host. Methods and Results: The method described herein is a polymerase chain reaction targeting the 16S rRNA gene of the Chlamydiaceae family, and the results demonstrate that the test reacts with five reference Chlamydiaceae but none of the 19 other bacterial species or five uninfected animal tissues tested. The results also indicate the enhanced sensitivity of this test when compared with conventional culture or serology techniques. This is demonstrated through parallel testing of six real clinical veterinary cases and confirmatory DNA sequence analysis. Conclusions, Significance and Impact of the Study: This test can be used by veterinary diagnostic laboratories for rapid detection of Chlamydiaceae in veterinary specimens, with no restriction of chlamydial species or animal host. The test does not differentiate chlamydial species, and if required, speciation must be carried out retrospectively using alternate methods. However, for the purpose of prescribing therapy for chlamydiosis, this test would be an invaluable laboratory tool.

Rapid and sensitive detection of Avibacterium paragallinarum in the presence of other bacteria using a 5' Taq nuclease assay: A new tool for diagnosing infectious coryza

A 5' Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5' Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5' Taq nuclease assay was demonstrated.

Laboratory studies on Australian isolates of Metarhizium anisopliae as a biopesticide for the cattle tick Boophilus microplus

Thirty-one isolates of Metarhizium anisopliae were bioassayed against the cattle tick (Boophilus microplus). More than half of the isolates showed a high degree of virulence to ticks. Radial growth curves for growth between 20 °C and 40 °C were obtained for all isolates. This information together with information on virulence will be important for the selection of isolates suitable to kill ticks on the surface of cattle. A biopesticide for cattle ticks must kill ticks rapidly at temperatures within the upper end of most isolates' growth curves. It was also found that the time taken to achieve 100% tick mortality in vitro using a virulent isolate could be halved by applying conidia in a 10% oil emulsion. Scanning electron microscopy and light microscopy were used to investigate and compare the germination and penetration of conidia formulated in aqueous and oil formulations. It was found that conidia in both formulations were able to germinate and produce appressoria on the surface of ticks in less than 11 h. Marked weakness within 26 h, followed by extensive hyphal growth on the cuticle characterised the invasion of ticks by M. anisopliae.

Diagnosis of a naturally occurring dual infection of layer chickens with fowlpox virus and gallid herpesvirus 1 (infectious laryngotracheitis virus).

An outbreak of acute respiratory disease in layers was diagnosed as being of dual nature due to fowlpox and infectious laryngotracheitis using a multidisciplinary approach including virus isolation, histopathology, electron microscopy and polymerase chain reaction (PCR). The diagnosis was based on virus isolation of gallid herpesvirus 1 (GaHV-1) in chicken kidney cells and fowlpox virus (FWPV) in 9-day-old chicken embryonated eggs inoculated via the chorioallantoic membrane. The histopathology of tracheas from dead birds revealed intra-cytoplasmic and intra-nuclear inclusions suggestive of poxvirus and herpesvirus involvement. The presence of FWPV was further confirmed by electron microscopy, PCR and histology. All FWPV isolates contained the long terminal repeats of reticuloendotheliosis virus as demonstrated by PCR. GaHV-1 isolates were detected by PCR and were shown to have a different restriction fragment length polymorphism pattern when compared with the chicken embryo origin SA2 vaccine strain; however, they shared the same pattern with the Intervet chicken embryo origin vaccine strain. This is a first report of dual infection of chickens with GaHV-1 and naturally occurring FWPV with reticuloendotheliosis virus insertions. Further characterization of the viruses was carried out and the results are reported here.

Comparative studies on the invasion of cattle ticks (Rhipicephalus (Boophilus) microplus) and sheep blowflies (Lucilia cuprina) by Metarhizium anisopliae (Sorokin).

Microscopic investigations over time were carried out to study and compare the pathogenesis of invasion of ticks and blowflies by Metarhizium anisopliae. The scanning electron microscope and stereo light microscope were used to observe and record processes on the arthropods' surfaces and the compound light microscope was used to observe and record processes within the body cavities. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle, particularly the outer endocuticle. Although large numbers of conidia adhered to the surface of blowflies, no conidia were seen to germinate on external surfaces. A single germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggests that fungal invasion of the blowfly is primarily through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on tick cuticles, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16.

Study of the structure and function of the sheath of tropically adapted bulls and risk factors for preputial eversion and preputial prolapse

Preputial prolapse is an obvious condition affecting bulls from many breeds. Unfortunately, the losses in production and welfare concerns associated with preputial prolapse can remain undetected for long periods of time in the extensive beef areas of northern Australia where the bulls are not inspected regularly. Thus, there is a critical need to identify the structural factors predisposing to preputial prolapse in young bulls so that they can be culled early. Despite there being no firm scientific evidence of an association between preputial eversion and preputial prolapse, it seems logical that the increased exposure of the sensitive prepuce as a consequence of preputial eversion may increase the risk of bulls developing preputial pathology, in particular preputial prolapse. This may be particularly relevant in Bos indicus bulls as they have a more pendulous sheath and thus eversion of the prepuce may be associated with a greater risk of injury to the prepuce compared to that in Bos taurus bulls. Further, studies of preputial eversion in Bos taurus bulls have concluded that there is an association between polledness and increased prevalence and severity (length of everted prepuce and duration of eversion) of preputial eversion due primarily to the absence or poor development of the caudal preputial muscles. No similar definitive work in Bos indicus bulls has been conducted and thus anatomical studies reported in this thesis were conducted to determine if a similar association occurred in Bos indicus bulls. A survey of a sample of large beef breeding herds in northern Australia found that preputial prolapse is a significant problem in Bos indicus and Bos indicus derived bulls and affected both young and older bulls. The importance of preputial prolapse confirmed the value of further research into the causes of this problem. A series of anatomical studies confirmed that preputial eversion in Bos indicus derived bulls was not more prevalent in polled bulls than horned bulls and was not associated with deficiency of the caudal preputial muscles as was established in Bos taurus bulls. An anatomical study of Bos indicus derived bulls with preputial prolapse found that preputial prolapse occurred in horned bulls of varying ages and these bulls did not have any evidence of deficiency in the caudal preputial muscles. However, preputial prolapse was observed in young polled bulls that had poorly developed or absent caudal preputial muscles. It was concluded that deficiency of the caudal preputial muscles in polled Bos indicus derived bulls may predispose to preputial prolapse at an early age, but no predisposing anatomical factors were found for horned Bos indicus derived bulls. In these studies, preputial eversion and preputial prolapse were found in horned Bos indicus derived bulls that did not have any preputial muscle deficiency and it was noted that preputial eversion was not related to the length of the prepuce. Further studies confirmed that preputial eversion was linearly and consistently associated with position of the glans penis within the sheath in Bos indicus derived bulls, and movement of the glans penis towards the preputial orifice consistently resulted in preputial eversion in these bulls. A method to objectively measure the relationship between movement of the glans penis within the sheath and preputial eversion was developed. Studies in humans have linked function of some abdominal muscles to function of the pelvic organs. This relationship was investigated in Bos indicus derived bulls to determine whether the function of specific abdominal muscles affected position of the penis in the sheath. Using the method developed to objectively measure the relationship between penis movement and preputial eversion, the abdominal muscles that potentially were associated with movement of the glans penis or preputial eversion were examined but no significant relationships were observed. In the anatomical study of Bos indicus derived bulls not affected with preputial prolapse a more pendulous sheath was associated with increased prevalence of preputial eversion. This relationship was confirmed for horned and polled bulls in the penis movement studies. Bos indicus derived bulls with more pendulous sheaths evert their prepuces more than bulls with less pendulous sheaths thus increasing the risk of damage to the prepuce either from the environment, other bulls, or from them inadvertently stepping on the everted prepuce when they get to their feet. Culling Bos indicus derived bulls with more pendulous sheaths should reduce the incidence of preputial eversion and possibly preputial prolapse. The anatomical study of Bos indicus derived bulls that did not have preputial prolapse demonstrates that there are herds of bulls where the polled bulls do not have any evidence of deficiency of the caudal preputial iv muscles. There is a need to develop a practical and cost effective test to identify polled Bos indicus bulls that have a deficiency in their caudal preputial muscles.

A field investigation into a suspected outbreak of pyrrolizidine alkaloid toxicosis in horses in western Queensland

A disease outbreak investigation was conducted in western Queensland to investigate a rare suspected outbreak of pyrrolizidine alkaloid (PA) toxicosis in horses. Thirty five of 132 horses depastured on five properties on the Mitchell grass plains of western Queensland died in the first six months of 2010. Clinical–pathological findings were consistent with PA toxicosis. A local variety of Crotalaria medicaginea was the only hepatotoxic plant found growing on affected properties. Pathology reports and departure and arrival dates of two brood mares provided evidence of a pre wet season exposure period. All five affected properties experienced a very dry spring and early summer preceded by a large summer wet season. The outbreak was characterised as a point epidemic with a sudden peak of deaths in March followed by mortalities steadily declining until the end of June. The estimated morbidity (serum IGG > 50 IU/L) rate was 76%. Average crude mortality was 27% but higher in young horses (67%) and brood mares (44%). Logistic regression analysis showed that young horses and brood mares and those grazing denuded pastures in December were most strongly associated with dying whereas those fed hay and/or grain based supplements were less likely to die. This is the first detailed study of an outbreak of PA toxicosis in central western Queensland and the first to provide evidence that environmental determinants were associated with mortality, that the critical exposure period was towards the end of the dry season, that supplementary feeding is protective and that denuded pastures and the horses physiological protein requirement are risk factors.