A. Pijpers

The highly pathogenic avian influenza A (H7N7) virus epidemic in The Netherlands in 2003--lessons learned from the first five outbreaks.

Abstract Clinical signs and gross lesions observed in poultry submitted for postmortem examination (PME) from the first five infected poultry flocks preceding the detection of the primary outbreak of highly pathogenic avian influenza (HPAI) of subtype H7N7 during the 2003 epidemic in the Netherlands are described. The absence of HPAI from the Netherlands for more than 75 yr created a situation in which poultry farmers and veterinary practitioners did not think of AI in the differential diagnosis as a possible cause of the clinical problems seen. Increased and progressive mortality was not reported to the governmental authorities by farmers or veterinary practitioners. It took 4 days from the first entry of postmortem material to notify the governmental authorities of a strong suspicion of an AI outbreak on the basis of a positive immunofluoresence test result. The gross lesions observed at PME did not comply with the descriptions in literature, especially the lack of hemorrhagic changes in tissues, and the lack of edema and cyanosis in comb and wattles is noted. The following lessons are learned from this epidemic: a) in the future, increased and progressive mortality should be a signal to exclude AI as cause of disease problems on poultry farms; b) intensive contact between the veterinary practitioner in the field and the veterinarian executing PME is necessary to have all relevant data and developments at ones disposal to come to a conclusive diagnosis; c) in an anamnesis, reporting of high or increased mortality should be quantified in the future (number of dead birds in relation to the number of birds brought to the farm to start production, together with the timing within the production cycle), or else this mortality cannot be interpreted properly; d) if clinical findings such as high mortality indicate the possibility of HPAI, the pathologist should submit clinical samples to the reference laboratory, even if PME gives no specific indications for HPAI; e) the best way to facilitate early detection of an HPAI outbreak is to have the poultry farmer and/or veterinary practitioner immediately report to the syndrome-reporting system currently in operation the occurrence of high mortality, a large decrease in feed or water intake, or a considerable drop in egg production; f) in order to detect low pathogenic avian influenza infections that could possibly change to HPAI, a continuous serologic monitoring system has been set up, in which commercial poultry flocks are screened for antibodies against AI virus of subtypes H5 and H7.

Comparison of Culture, PCR, and Different Serologic Tests for Detection of Mycoplasma gallisepticum and Mycoplasma synoviae Infections

Abstract In this study, the technical performance of culture, two commercially available polymerase chain reaction (PCR) tests, rapid plate agglutination (RPA) test, hemagglutination inhibition (HI) test, and eight commercially available enzyme-linked immunosorbent assays (ELISAs) were compared for the detection of avian mycoplasma infections from 3 days postinfection (d.p.i.) through 35 d.p.i. The tests were carried out on samples from specified pathogen-free layers that were infected at 66 wk of age with recent Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) field strains, MS and MG ATCC strains, and Mycoplasma imitans (MIM), respectively. Results showed a high percentage of positive samples in the homologous infected groups and a high percentage of negative samples (100%) in the uninfected and heterologous infected groups during 35 d.p.i. of both culture and PCR tests. For the group infected with the MG 15302 ATCC strain, serology was more sensitive than bacteriology. All MG and MS tests, with the exception of MG ELISA kit D showed a lower percentage of positive samples during 35 d.p.i. for the detection of the MG and MS ATCC strain infection compared with that of the field strains. Also, the number of cross-reactions (false positives) in the serologic tests was lower after infection with an ATCC strain than after an infection with the MG or MS field strain. Contradictory to other studies, the ELISAs and the RPA test using undiluted serum showed a relatively high number of false-positive results. The MG ELISAs (except ELISA kit D) showed more false-positive results (up to 37%) in the MIM-infected group than in the MS-infected groups. This was not unexpected, as MIM and MG have a close antigenic relationship. The results of the serologic tests in this study showed that a certain level of false-positive results can be expected in about any serologic test. Although the level of false-positive results varied between several serologic tests, this study showed that it is not advisable to rely completely on one test (system) only.

An observational study of Eimeria species in housed cattle on Dutch dairy farms.

The prevalence of oocysts of Eimeria species in calves (n = 334), yearlings (n = 254) and cows (n = 1314) was determined on 38 Dutch dairy farms. Twelve species of Eimeria were identified in faecal specimens by sucrose-flotation. The prevalences of Eimeria spp. differed markedly in the different age classes on individual farms as well as between farms. The overall prevalence of Eimeria oocysts in faecal specimens was 46% for calves, 43% for yearlings and 16% for cows. The number of oocysts excreted was generally low in cows and yearlings, whereas high numbers of oocysts per gram of faeces (OPG) were exclusively observed in calves. No cases of clinical coccidiosis were observed in this survey. Linear regression analysis showed that there is significant reduction in the OPG levels (P < 0.05) in calves infected with Eimeria, aged between 7 and 38 weeks. Finally, the data are discussed in relation to management practices and the acquisition of immunity.

Comparison of methods for in vitro testing of susceptibility of porcine Mycoplasma species to antimicrobial agents.

The MICs of 18 antimicrobial agents used against strains of three porcine Mycoplasma species were determined by a serial broth dilution method. Twenty field strains of M. hyorhinis, ten field strains of M. hyopneumoniae, six field strains of M. flocculare, and the type strains of these species were tested. Twelve field strains and the type strain of M. hyorhinis were also tested by an agar dilution method. Tests were read at various time points. When the broth dilution method was used, the final MIC had to be read 2 days after color changes had stopped. MICs of tetracycline, oxytetracycline, doxycycline, and minocycline were low for the three Mycoplasma species tested. MICs of chlortetracycline were 8 to 16 times higher than MICs of the other tetracyclines. Spiramycin, tylosin, kitasamycin, spectinomycin, tiamulin, lincomycin, and clindamycin were effective against all strains of M. hyorhinis and M. hyopneumoniae. The quinolones were highly effective against M. hyopneumoniae but less effective against M. hyorhinis. The susceptibility patterns for M. hyopneumoniae and M. flocculare were similar.

The prevalence of patent lungworm infections in herd of dairy cows in the Netherlands

The results of a survey on the prevalence of patent lungworm infections in herds of dairy cows in the Netherlands are presented. Low patent infections were recorded in February-March on six out of 40 farms in at least one out of 40 cows. Between mid-April and mid-June low patent infections were detected on 28 out of 39 of these farms in one to four of 40 cows. Two farms on which cows were positive in the first round were negative in the second round. One to three positive cows were found on six out of a total of 15 farms revisited in July-August. These results show that lungworm infections are cycled within herds of dairy cows in the Netherlands at a low level. This indicates that dairy cows are important as carriers for lungworm, particularly in spring. The increased patency of lungworm in cows from winter to spring may be explained by maturation of inhibited larvae.

Selective effects of a bacterial infection (Actinobacillus pleuropneumoniae) on the hepatic clearances of caffeine, antipyrine, paracetamol, and indocyanine green in the pig

1. In order to investigate the effect of a bacterial acute phase response model on drug disposition in vivo, plasma clearances of antipyrine, caffeine, paracetamol and indocyanine green were investigated in the healthy and Actinobacillus pleuropneumoniae-infected pig. 2. Indocyanine green plasma and endogenous creatinine clearance were not changed during the infection, which indicates that hepatic blood flow and renal function were not significantly affected. 3. In the A. pleuropneumoniae-infected pig, plasma clearances of antipyrine and caffeine, both marker substrates for hepatic oxidative biotransformation, were decreased by 72 and 68% respectively. The clearance of paracetamol, a drug mainly glucuronidated in the pig, was reduced by 39%. 4. It is concluded that the most important change in drug elimination during an acute phase response induced by A. pleuropneumoniae is a suppression of oxidative hepatic biotransformation.

Experimental reproduction of malabsorption syndrome with different combinations of reovirus, Escherichia coli and treated homogenates obtained from broilers

SUMMARY. Attempts to reproduce malabsorption syndrome (MAS) by oral inoculation with several different combinations including intestinal homogenate, reovirus, and hemolytic Escherichia coli obtained from MAS-affected chickens and intestinal homogenate from healthy chickens (healthy homogenate) were performed in 1-day-old specific-pathogen-free (SPF) broilers. The MAS homogenate, serving as a positive control, induced weight gain depression and intestinal lesions such as cystic crypts of Lieberkühn, villus atrophy, and lymphoid and/or granulocytic infiltration. The healthy homogenate, the formalin-treated MAS homogenate, the formalin-treated healthy homogenate, and phosphate-buffered saline caused neither weight gain depression nor intestinal lesions. We were able to reproduce both weight gain depression and intestinal lesions by inoculation of reovirus either combined with the formalin-treated MAS homogenate or combined with healthy homogenate. Surprisingly, when hemolytic E. coli was added to the combination of reovirus with formalin-treated MAS homogenate, this did not cause weight gain depression although this combination caused the described intestinal lesions. Identical results were obtained with the combination of formalin-treated MAS homogenate with hemolytic E. coli or the combination of reovirus with hemolytic E. coli. The intestinal lesions were more severe and developed faster by combinations including reovirus and formalin-treated MAS homogenate. This study indicates that a combination of enteropathogenic reovirus with other agents or substances that are present in an intestinal homogenate from MAS-affected and healthy chickens can induce MAS in SPF broilers. Escherichia coli is not essential for induction of weight gain depression but can play a role in development of intestinal lesions. Furthermore, intestinal lesions alone will not always result in weight gain depression.

Transmission of infectious bronchitis virus within vaccinated and unvaccinated groups of chickens

The aim of this study was to determine whether vaccination against infectious bronchitis virus (IBV) reduces virus transmission, i.e. to test whether IBV transmission among vaccinated chickens is significantly reduced compared to that among unvaccinated chickens. In two vaccinated and two unvaccinated groups of SPF chickens, a standard measure for virus transmission, the reproduction ratio (R) was determined. R is defined as the average number of new infections caused by one typical infectious individual during its entire infectious period. A single vaccination by eye-drop with IBV H120 reduced the transmission of the IBV challenge virus among the vaccinated chickens (estimated R = 0.69, s.e. = 0.33) significantly (P < 0.05) compared to the transmission among the unvaccinated chickens (estimated R = 19.95, s.e. = 12.41). The possible implications for further study, including selection or development of vaccines are discussed.

Intravenous catheterisation of conventional pigs without application of antimicrobial agents

In six experiments 43 castrated male conventional pigs weighing 25-41 kg were catheterised by inserting a cannula via the jugular vein into the cranal caval vein. The catheters were taped to the spinal neck region where the tap stops were located. Antimicrobial agents were not applied. One pig died 32 hour after surgery from Porcine Stress Syndrome. The catheters remained patent for at least nine days in 38 of the remaining 42 animals (90%). In two animals the catheter by mistake was not inserted into the jugular vein. Two animals got catheters with a one-way blockage four days after surgery. In these animals autopsy revealed thrombosis and phlebitis of the occluded vein and a valve-like thrombus at the tip of the catheters. In seven of the 43 pigs the effects of anaesthesia, surgery and catherisation were followed using rectal temperature and haematological and some blood biochemical parameters for nine days after the surgery. It is concluded that this catheterisation technique, without application of antimicrobial agents, can be used well for experimental infections and pharmacokinetic studies.

Dose-dependent pharmacokinetic interaction between antipyrine and paracetamol in vivo and in vitro when administered as a cocktail in pig

1. The pharmacokinetic interactions between paracetamol (PA) and antipyrine (AP) were studied in pigs in order to investigate the usefulness of this combination for the simultaneous assessment of oxidative and conjugative metabolism. 2. When both drugs were given at a dose of 5 mg/kg, AP plasma clearance was reduced from 2.22 to 0.96 lh-1 kg-1. PA clearance was not changed in comparison with control values. 3. At a dose of 2 mg/kg no pharmacokinetic interaction between the two drugs was observed. 4. The only oxidative AP metabolite found in urine was 4-hydroxyantipyrine (4-OHA). It accounted for 80% of the dose and, like PA, it was completely glucuronidated. 5. The glucuronidation of PA has been studied in vitro in pig liver microsomes. The apparent Km value for PA glucuronidation was 40 mM with a Vmax = 54 nmol min-1 mg protein-1. To determine if 4-OHA and PA competed for the same UDP-glucuronosyl-transferase, the effect of 4-OHA and AP on PA glucuronidation was studied. It appeared that 4-OHA was a competitive inhibitor with a Ki app = 0.07 microM, whereas AP had no effect. 5. Results suggest a dose-dependent interaction between AP and PA, which may be due to competition at the level of glucuronidation. Therefore, the usefulness of AP and PA in vivo in a cocktail for metabolism studies is limited.