Peter Lind

Interpretation of the Gamma Interferon Test for Diagnosis of Subclinical Paratuberculosis in Cattle

ABSTRACT A group of 252 cattle without clinical signs of paratuberculosis (paraTB) in 10 herds infected with paraTB and a group of 117 cattle in 5 herds without paraTB were selected. Whole-blood samples were stimulated with bovine, avian, and johnin purified protein derivative (PPD) and examined for gamma interferon (IFN-γ) release. For diagnosis of paraTB, satisfactory estimated specificities (95 to 99%) could be obtained by johnin PPD stimulation irrespective of interpretation relative to bovine PPD or no-antigen stimulation alone, but numbers of test positives in the infected herds varied from 64 to 112 with different interpretation criteria. For a limited number of test-positive animals, no change in the test results could be observed with increasing antigen concentrations but IFN-γ responses were significantly reduced (P < 0.0001) and four out of seven reactors tested negative when stimulation was performed on day-old samples. Denmark is free of bovine tuberculosis, but cross-reactivity with paraTB could be documented for cattle more than 14 months old in paraTB-infected herds compared with those in non-paraTB-infected herds. In both paraTB-free and paraTB-infected herds, false positives were observed when the test was applied to calves less than 15 months of age. Until novel antigen formulations more specific for these diseases are available, interpretation of the IFN-γ test must be individually adjusted to fit specific needs and the context within which the test is applied and, for paraTB, the test seems most appropriate for use as a supportive tool for evaluation of disease-preventive measures in young stock.

THE TIME COURSE OF THE SPECIFIC ANTIBODY RESPONSE BY VARIOUS ELISAS IN PIGS EXPERIMENTALLY INFECTED WITH TOXOPLASMA GONDII

With the aim of developing routine serological tests for monitoring the Toxoplasma infection status of Danish swine herds, four ELISAs based on tachyzoite antigen were set up: (1) an indirect ELISA for IgG-antibody; (2) a blocking ELISA for antibody to the membrane antigen, P30; (3) an indirect ELISA for IgM; (4) a reverse, antibody-catching IgM-ELISA. Groups of pigs (number between 6 and 10) were inoculated with tachyzoites of the RH-strain, tissue cysts of two complete strains, or oocysts in two doses (10(3) and 10(4). All inoculations were tolerated well. Irrespective of strain and stage used for inoculation, specific IgG and anti-P30 blocking activity appeared after 1-2 weeks, with OD-values stabilizing after 3-6 weeks and persisting throughout the study period (3-4 months). Specific IgM appeared quickly, but was short-lived (approximately 2 weeks). A cut-off OD-value of 0.36 for positive seroreaction in the indirect IgG-ELISA was determined on the basis of 69 sera from four herds, investigated in the dye-test (serum dilution 1:10) and ELISA. The chosen cut-off gave optimal combined sensitivity and specificity of 0.94 and 0.92, respectively, using the dye-test as a standard. Corresponding figures for the blocking ELISA were 37% inhibition as cut-off, with sensitivity and specificity of 0.94 and 0.94, respectively. Sera from a total of 87 pigs, experimentally infected with bacteria of the genera Salmonella, Yersinia or Actinobacillus and with the parasites Isospora suis, Trichinella spiralis or Ascaris suum, in no case produced cross-reactions in the IgG-ELISA. However, 3/9 pigs inoculated with 50 000 sporocysts of Sarcocystis miescheriana gave maximal OD-readings of 0.40-0.45 during the 13-15 weeks observation period. None of the sera from heterologously infected animals produced inhibitions in the anti-P30 blocking ELISA exceeding 36%.

Cryptosporidium andersoni from a Danish cattle herd: identification and preliminary characterisation

In November 1997, Cryptosporidium andersoni, for the first time, was isolated from a Danish heifer. The isolate was characterised morphologically, molecularly, and furthermore inoculated into mice and one calf. Data on the distribution of cryptosporidia in the herd of origin were obtained at two separate visits in December 1997 and April 1998. C. andersoni was detected in 27 (19.0%) of 142 cattle examined at the first visit, whereas C. parvum was found in six (4.2%). At the following visit 42 (28.0%) of 150 cattle excreted C. andersoni, while 25 (16.7%) were positive for C. parvum. Oocysts of the Danish C. andersoni isolate were ovoid, 7.3(6.5-8.0) x 5.7(5.0-7.0) microm(2) (n=25), with smooth, colourless, single layer oocyst wall and distinct oocyst residuum. The length to width ratio was 1.27 (1.14-1.40, n=25). The identification was verified by sequencing of a 246bp fragment of the rDNA, which was identical to Cryptosporidium muris, the calf genotype (AF093496). The Danish C. andersoni isolate was not transmissible to mice, whereas oocysts were detected in the faeces of one experimentally infected calf from 25 days post-infection (DPI) and shed intermittently at low numbers until 165 DPI, the day of euthanasia. No macroscopic or microscopic changes that could be attributed to infection with C. andersoni were seen in the gastro-intestinal tract of the experimentally infected calf following necropsy and histological examination. This is to our knowledge the first report of C. andersoni in Scandinavia.

Trichinella spiralis, T. britovi, and T. nativa : infectivity, larval distribution in muscle, and antibody response after experimental infection of pigs

Abstract The infectivity of Trichinella spiralis, T. nativa, and T. britovi was experimentally compared in pigs. Blood sampling was performed weekly, and muscle juices were obtained at slaughter 10 weeks after inoculation. Muscle larvae were found in all of four pigs inoculated with T. spiralis [mean 190 larvae per gram (lpg)] and in three of four pigs inoculated with T. britovi (mean 7 lpg). No larvae were found in pigs inoculated with T. nativa. For T. spiralis and T. britovi, the neck muscle (m. splenius) appears to be a predilection site in addition to the tongue, the diaphragm, and the jaw. High antibody responses were found in all experimental groups, independent of the antigen used, and even in pigs in which no muscle larvae were recovered. The strong and consistent antibody response found with meat juice indicates the usefulness of this material where a blood sample is not obtainable, e.g. meat samples from wild animals. Immunoblotting (Western blots) on slaughter sera revealed no species specificity when comparing homologous versus heterologous staining.

Response to repeated inoculations withAscaris suum eggs in pigs during the fattening period

This experimental study on pigs was designed to simulate natural, long-term exposure toAscaris suum under modern management conditions. Parasite kinetics were followed in pigs receivingA. suum eggs as repeated trickle inoculations at two dose levels beginning at a body weight of 25kg until their slaughter at 90kg (baconers). In pigs inoculated twice weekly with 500 eggs, there was an initial marked rise in the numbers of hepatic milk spots, but as early as around week 6 after the start of inoculations and until week 16, at which time the last pigs were slaughtered, the numbers of spots diminished drastically. In pigs receiving only 25 eggs twice weekly, low and moderately fluctuating numbers of spots were seen throughout the experiment. Larvae recoverable from the livers and lungs were observed mainly during the beginning of the experiment. Before patency, immature intestinal worms were found in moderate numbers that showed a rough positive correlation with the dose levels, but at the time at which adult worms started to appear, immature parasites could practically no longer be found. In all, only 10 of 40 pigs harbored adults, and 4 of these 10 pigs harbored, 80% of the total worm population. The results show that acquired dosedependent host responses toA. suum play an important role in regulating the worm population along the migratory route of the parasite and that the final burden of worms in the small intestine is dose-independent and highly variable.This experimental study on pigs was designed to simulate natural, long-term exposure to Ascaris suum under modern management conditions. Parasite kinetics were followed in pigs receiving A. suum eggs as repeated trickle inoculations at two dose levels beginning at a body weight of 25 kg until their slaughter at 90 kg (baconers). In pigs inoculated twice weekly with 500 eggs, there was an initial marked rise in the numbers of hepatic milk spots, but as early as around week 6 after the start of inoculations and until week 16, at which time the last pigs were slaughtered, the numbers of spots diminished drastically. In pigs receiving only 25 eggs twice weekly, low and moderately fluctuating numbers of spots were seen throughout the experiment. Larvae recoverable from the livers and lungs were observed mainly during the beginning of the experiment. Before patency, immature intestinal worms were found in moderate numbers that showed a rough positive correlation with the dose levels, but at the time at which adult worms started to appear, immature parasites could practically no longer be found. In all, only 10 of 40 pigs harbored adults, and 4 of these 10 pigs harbored 80% of the total worm population. The results show that acquired dose-dependent host responses to A. suum play an important role in regulating the worm population along the migratory route of the parasite and that the final burden of worms in the small intestine is dose-dependent and highly variable.

Cytokine mRNA Profiles in Bronchoalveolar Cells of Piglets Experimentally Infected in Utero with Porcine Reproductive and Respiratory Syndrome Virus: Association of Sustained Expression of IFN-γ and IL-10 after Viral Clearance

An experimental model was used to investigate mRNA cytokine profiles in bronchoalvolar cells (BALC) from piglets, infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV). The BALCs were analyzed for the cytokines TNF-alpha, IFN-gamma, IL-8, IL-10, and IL-12(p40) by real-time TaqMan polymerase chain reaction in 2-, 4-, and 6-week-old piglets, respectively. High levels of IFN-gamma mRNA was detected in all piglets, while IL-10 was upregulated in 2-week-old piglets, was at normal levels in 4-week-old piglets, and elevated again in 6-week-old piglets. IL-12 was weakly elevated in all three age groups. Virus was reduced by 50% in 4-week-old piglets and cleared by 6 weeks of age. The sustained expression of IFNgamma and reduction of IL-10 production indicate an important role for these cytokines in immunity to PRRSV.

Non-lethal infection parameters in mice separate sheep Type II Toxoplasma gondii isolates by virulence

The zoonotic protozoan parasite Toxoplasma gondii can infect all warm-blooded animals, but virulence of isolates has previously been characterised mainly by the ability to kill mice after experimental infections. In the present study, 15 Type II strains of T. gondii, isolated from five adult sheep, six sheep abortions, two pigs, one cat and one fox were examined for their virulence to young mice by less dramatic parameters. Clinical disease of inoculated mice, directly evidenced by reduced weight gain, was correlated to increase in serum level of haptoglobin and level of specific antibodies. Although Type II T. gondii strains are non-virulent to mice by lethality studies, significant differences in mouse virulence were observed between the strains of T. gondii isolated either from adult sheep or from sheep abortions. It was not possible to characterise strains isolated from sheep abortions as being more or less virulent than strains isolated from adult slaughter sheep.

Molecular characterization of Danish Cryptosporidium parvum isolates.

The genetic polymorphism among 271 Danish Cryptosporidium isolates of human and animal origin was studied by partial amplification and sequencing of the Cryptosporidium oocyst wall protein (COWP) gene, the 1 8S rDNA, and a microsatellite locus. Furthermore, the microsatellite locus was studied directly using fragment analysis. A comparative analysis of DNA sequences showed the presence of 3 different subgenotypes (Cl, C2 and C3) in C. parvum isolates from Danish cattle, with prevalences of 16.7, 17.2 and 73.1% including 13 (7.0%) mixed infections. Subgenotype Cl was significantly more prevalent (P < 0.001) in the southern part of Denmark. In Cryptosporidium isolates of human origin the anthroponotic subgenotype H1 was identified, in addition to the zoonotic subgenotypes C1, C2, and C3. Of 44 human samples, 56.8% were anthroponotic, whereas 40.9% were zoonotic genotypes. One human isolate was characterized as C. meleagridis. The porcine Cryptosporidium isolates (N = 4) revealed a pattern which was genetically distinct from human and bovine isolates. Cryptosporidium in a hedgehog (Erinaceus europaeus L.) was identified for the first time. By microsatellite sequencing the hedgehog isolate showed a subgenotype distinct from the previously detected types. The assignment to subgenotype by microsatellite sequencing and fragment typing was 100% identical in samples where results were achieved by both methods. In addition, the fragment analysis proved more sensitive, easier, faster, and less expensive compared to sequencing.

Resistance to Ascaris suum in parasite naïve and naturally exposed growers, finishers and sows

Commercially reared growers, finishers, and sows of Danish Landrace x Yorkshire crossbred were inoculated orally with Ascaris suum at 50 eggs kg-1 body weight. White spots on the serosal surface of livers and total larval recoveries from lungs were recorded 7 days later. The response in pigs originating from a specific pathogen free and parasite free herd (parasite naïve) was observed in the three different age groups and compared with age-matched pigs from a herd maintained in a facility contaminated with A. suum (naturally exposed). The pre-inoculation immune status of the various groups was characterized serologically using antigen preparations derived from various stages of A. suum. Inoculation of all age groups of parasite naïve pigs with A. suum eggs produced relatively high liver white spots and lung larvae, although expression of these counts as a percentage of the inoculum showed a moderate age-related resistance from growers to finishers to sows. In contrast, pigs naturally exposed to A. suum expressed strong immunity to a challenge infection as few or no larvae were detected in the lungs. In addition, growers, finishers, and sows from the naturally exposed herd had significantly higher levels of serum IgG/IgA to several different A. suum antigens compared with pigs from the parasite nave herd. Liver white spots, expressed as a percentage of the inoculum, were highest in growers from the naturally exposed herd but were markedly reduced in finishers and sows from that herd. In fact, few or no white spots were observed in naturally exposed sows, while sows from the parasite-naïve herd had in excess of 300 liver white spots following challenge. These results indicate that commercially raised pigs that are exposed to A. suum develop a strong protective immunity that ultimately produces a complete pre-hepatic barrier to larval migration, while pigs raised parasite free remain susceptible to infection.

Cytokine Profiles in Peripheral Blood Mononuclear Cells and Lymph Node Cells from Piglets Infected In Utero with Porcine Reproductive and Respiratory Syndrome Virus

ABSTRACT The aim of the present study was to investigate at 2, 4, and 6 weeks after birth cytokine expression by peripheral blood mononuclear cells and bronchial lymph node cells from piglets infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV). Technically, by flow cytometry we were able to measure gamma interferon (γ-IFN), tumor necrosis factor alpha (TNF-α), interleukin-4 (IL-4), and IL-8 levels. In general, we found increases in the percentages of IL-4-, γ-IFN-, and TNF-α-producing lymphocytes in the infected piglets compared to the percentages in the uninfected control animals, while there was a decrease in the percentage of IL-8-producing monocytes. We believe that these findings reflect a general lymphocyte activation stage that is created due to the infection and that occurs in combination with impairment of the monocyte function, possibly due to the ongoing viral replication in these cells. Single-cell bronchial lymph node preparations exhibited very much the same cytokine profiles as peripheral blood mononuclear cells except for a lack of IL-8 production. When the levels of the individual cytokines in the three groups of PRRSV-infected piglets were compared, the levels of cytokine expression at 4 weeks diverged from those at 2 and 6 weeks, in that there was a significant decrease in the numbers of lymphocytes producing γ-IFN and TNF-α. This tendency was also observed among blood monocytes and lymph node macrophages. Possible reasons for this temporary immunosuppression in the piglets at 4 weeks are discussed.