Infectivity and virulence in a serodeme of Trypanosoma vivax

The course of infection by a mouse-adapted Trypanosoma vivax was studied. Serological variants were isolated from sheep and their infectivity and virulence were studied in mice and sheep. Isolates obtained in the early stage of the infection were highly virulent while isolates obtained at the terminal stage of the infection were found to be extremely virulent in both mice and sheep. Trypanosoma vivax organisms isolated in between these series of parasitaemias were of low to moderate virulence in both groups of animals. The degree of virulence exhibited by each isolate was closely related to the viability of the organisms. The success of making clones was equally found to be related to the virulence of the isolates.     

Comparative pathogenicity of three genetically distinct types of Trypanosoma congolense in cattle: clinical observations and haematological changes

The pathology of African bovine trypanosomosis was compared in Zebu cattle subcutaneously inoculated with three clones of trypanosomes corresponding to the three genetically distinct types of Trypanosoma congolense; savannah-type, west African riverine/forest-type and kilifi-type. All inoculated animals became parasitaemic between 7 and 11 days post-infection (dpi). The savannah-type showed consistently higher levels of parasitaemia and lower packed red cell volume percentages and leukocyte counts than the other two types. The syndrome was also more severe in the savannah-type and led inexorably to death between 29 and 54 dpi while animals with the forest or the kilifi-types recovered from earlier symptoms and haematological alterations after 3 months of infection. By the end of the experiment, the animals self-cured from the forest-type infection and the kilifi-type passed under control. The results of the present study indicated clear difference in pathogenicity between the three types of T. congolense; the savannah-type was virulent while the forest-type was of low pathogenicity and the kilifi-type was non-pathogenic.     

Evaluation of virulence of Mycoplasma hyopneumoniae field isolates

The course of enzootic pneumonia, caused by Mycoplasma hyopneumoniae, is strongly influenced by management and housing conditions. Other factors, including differences in virulence between M. hyopneumoniae strains, may also be involved. The aim of this study was to evaluate the virulence of six M. hyopneumoniae field isolates and link it to genetic differences as determined by randomly amplified polymorphic DNA (RAPD) analysis. Ninety, conventional M. hyopneumoniae-free piglets were inoculated intratracheally with the field isolates, a virulent reference strain or sterile culture medium. Animals were examined daily for the presence of disease signs and a respiratory disease score (RDS) was assessed per pig. Twenty-eight days post infection, pigs were euthanized, blood sampled and a lung lesion score was given. Lung samples were processed for histopathology, immunofluorescence testing for M. hyopneumoniae and isolation of M. hyopneumoniae. RAPD analysis was performed on all M. hyopneumoniae strains. Significant differences between isolates were found for the RDS, lung lesion score, histopathology, immunofluorescence and serology. Based on the results of the different parameters, isolates were divided into three "virulence" groups: low, moderately and highly virulent strains. Typically, a 5000 bp RAPD fragment was associated with the highly and moderately virulent strains whereas it was absent in low virulent strains. It was concluded that high variation in virulence exists between M. hyopneumoniae strains isolated from different swine herds. Further studies are required to determine whether the 5000 bp fragment obtained in the RAPD analysis can be used as a virulence marker.     

Trypanosome infections in warthogs (Phacochoerus aethiopicus) in the Gambia.

The prevalence of trypanosome infections in warthogs (Phacochoerus aethiopicus) in The Gambia was found to be 11% of a sample of 62 animals. All isolates were identified as Trypanosoma simiae. Serological evidence indicated a higher level of exposure to T. simiae, but results were inconclusive for the presence of Trypanosoma congolense. The course of T. simiae infection in warthog piglets showed a rapidly rising parasitaemia, with a concomitant fall in packed cell volume, and resulted in a prolonged period of low-level parasitaemia. The same infections killed domestic piglets.     

Identification of unique DNA sequences present in highly virulent 2009 Alabama isolates of Aeromonas hydrophila

In 2009, a disease outbreak caused by Aeromonas hydrophila occurred in 48 catfish farms in West Alabama, causing an estimated loss of more than 3 million pounds of food size channel catfish. Virulence studies have revealed that the 2009 isolates of A. hydrophila are at least 200-fold more virulent than a 1998 Alabama isolate AL98-C1B. However, up to now, no molecular markers have been identified to differentiate the highly virulent 2009 isolates from other isolates of A. hydrophila. To understand the genetic differences between the highly virulent 2009 isolates and the less virulent AL98-C1B at molecular level, PCR-select bacterial genome subtractive hybridization was used in this study. A total of 96 clones were selected from the subtractive genomic DNA library. Sequencing results revealed that the 96 clones represented 64 unique A. hydrophila sequences. Of the 64 sequences, three (hypothetical protein XAUC_13870, structural toxin protein RtxA, and putative methyltransferase) were confirmed to be present in the three virulent 2009 Alabama isolates but absent in the less virulent AL98-C1B. Using genomic DNAs from nine field isolates of A. hydrophila with different virulence as templates, two sequences (hypothetical protein XAUC_13870 and putative methyltransferase) were found to be only present in highly virulent A. hydrophila isolates, but absent in avirulent isolates.     

Studies on the influence of non-steroidal anti-inflammatory drugs upon trypanosomiasis in goats and sheep

The non-steroidal anti-inflammatory drug (NSAID) flurbiprofen caused a rise in parasitaemia in goats infected with Trypanosoma vivax, Trypanosoma congolense and Trypanosoma brucei. All trypanosome-infected goats treated with flurbiprofen showed many dividing trypanosomes. This also included the short-stumpy forms of T. brucei. In T. vivax-infected goats flurbiprofen treatment resulted in 100% mortality in the acute and chronic stages of the infection. The increase in parasitaemia of T. brucei infected goats, treated with flurbiprofen, was not associated with an increase in mortality. The increase in parasitaemia of T. congolense-infected goats, treated with flurbiprofen, tended to be associated with a somewhat higher mortality but this was statistically not significant. The significant rise in parasitaemia could be reproduced in T. brucei-infected sheep without, however, killing the animals. Two other NSAIDs were also studied. Suprofen caused a rise in parasitaemia and 100% mortality when given to goats in the acute stage of T. vivax infection. Results with flunixin meglumine, when tested in T. brucei infected goats, were not conclusive.     

Recirculation of Trypanosoma brucei brucei in cattle after T. congolense challenge by tsetse flies

The effect of challenging cattle, chronically infected with Trypanosoma brucei brucei, with T. congolense on the development of the T. b. brucei infection was investigated. For this purpose, nine experimental animals were first infected with T. b. brucei through the bites of infected tsetse flies. Once the T. b. brucei had developed into a chronic infection, that was difficult to detect using routine parasitological diagnostic tools, seven of the experimental animals were challenged by tsetse flies infected with T. congolense. Two of the animals infected with T. b. brucei were kept as control. The infection with T. congolense resulted in a sudden increase in the parasitaemia of T. b. brucei. In the T. b. brucei control animals, on the other hand, the parasitaemia remained below the level of detection. The epidemiological repercussions of this increase in the parasitaemia of T. b. brucei after infection with T. congolense are discussed.     

The role of antibody in natural resistance to African trypanosomiasis

The nature and usefulness of trypanoresistance in West African Baoule cattle was studied by exposing animals in areas of high Glossina density. Under such conditions, Zebu and some Baoule died soon with high parasitaemia while resistant Baoule showed little patent parasitemia, almost no anaemia and thrived. Subsequently the role that specific antibody may play in such trypanoresistance was analyzed. In the first instance we examined protective antibody titres during T. congolense infection of inbred mice strains, comparing results obtained in trypanosome-mouse combinations where the host controls parasitemia and survives with those obtained when the host fails to control parasitemia and dies. We then attempted to extend these observations to cattle by following the disease course and appearance of neutralizing antibodies in animals of known sensitivity to natural Glossina challenges, following artificial challenge with T. congolense infected Glossina.     

The pathophysiology of ovine trypanosomosis: haematological and blood biochemical changes.

The course of Trypanosoma congolense infection in sheep was followed for 96 days. Infected animals developed fluctuating parasitaemia, macrocytic normochromic anaemia and leucocytosis which was principally a lymphocytosis. Following treatment with the trypanocidal drug, diminazene aceturate at 84 days after infection, the haematological values returned to normal within 12 days. Infected sheep developed hypocholesterolaemia and hypophospholipidaemia leading to a reduction in total serum lipids. This study has shown that sheep infected with T. congolense develop anaemia, the onset of which follows the first wave of parasitaemia. The changes in blood lipids observed in infected sheep appeared to be related to the intensity and duration of parasitaemia.     

Protein variability among Mycoplasma hyopneumoniae isolates

Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the protein variability of Mycoplasma hyopneumoniae isolates. Fifty-six M. hyopneumoniae isolates from 6 different countries and 37 different herds were used. From eight herds, more than one isolate was available. All SDS-PAGE patterns of isolates originating from different herds were clearly divergent. Intra-species protein variability was quantified using the reference strain J and seven field strains all obtained from different herds and classified according to virulence. Between the field strains, a variability of 25% was found, while the culture-adapted strain J was clearly divergent and showed 30% variability with the field strains. No clustering according to virulence was obtained, but a protein band of about 181 kDa was present in the two highly virulent isolates whereas this protein band was absent in the moderately and low virulent isolates. Protein patterns of isolates derived from different animals from the same herd, were identical or differed in only a few protein bands. This study clearly indicates that, in agreement with previous studies on genomic diversity of M. hyopneumoniae isolates, proteomic variability within the species is high. Our study did not find clear evidence that more than one M. hyopneumoniae isolate circulates within a herd at a specific time point. The minor differences found between M. hyopneumoniae isolates from the same herd might reflect the organism's ability to alter its proteomic expression profile under field conditions.     

Susceptibility of West African Dwarf goats and WAD x Saanen crosses to experimental infection with Trypanosoma congolense

West African Dwarf goats (WADs) and their Saanen crosses were experimentally infected with Trypanosoma congolense. No significant differences were found between trypanosome parasitaemia and antibody response of the crossbred and WAD goats. Neither the WAD goats nor the Saanen crosses were able to control the drop in PCV following trypanosome infection. The level of anaemia caused by the trypanosome infection was similar in the two breeds during the trial. Based on these findings, no difference in tolerance or susceptibility to T. congolense could be demonstrated between the WAD goats and their Saanen crosses. Although the weight of all goats increased during the trial, the crosses gained significantly more weight than the WAD goats. The trypanosome infection reduced the growth rate of both breeds, but this reduction was not statistically significant. Crossbreeding trypanotolerant WADs with trypanosusceptible Saanen goats might, therefore, be an effective means of increasing productivity.     

Complement resistance,as determined by viable count and flow cytometric methods,and its association with the presence of iss and the virulence of avian Escherichia coli

Previous work in our labs has shown that avian Escherichia coli virulence is correlated with resistance to complement. Also, our studies have revealed that the presence of the increased serum survival gene (iss), known to contribute to the complement resistance and virulence of mammalian E. coli, may predict the virulent nature of an avian E. coli isolate. This relationship warrants further research, but further clarification of the relationship among virulence, complement resistance, and iss sequences requires use of complement susceptibility assays. Such assays, unfortunately, are labor-intensive, expensive, and difficult to perform. In the present study, the results of two complement susceptibility assays for 20 E. coli isolates, 10 incriminated in avian colibacillosis and 10 from the intestinal tracts of apparently healthy birds, were compared in an attempt to determine if flow cytometric analysis was a reasonable alternative to a viable count assay. In addition, the virulence of these isolates for chick embryos was determined, and each isolate was examined for the presence of iss using amplification techniques. The flow cytometric method was found to be repeatable for most isolates, and its results showed moderate agreement with those obtained through viable counts. All intestinal isolates of healthy birds proved avirulent using the embryo lethality assay; however, not all isolates from sick birds were demonstrated to be virulent. Possible explanations of these results include that the methods originally used to isolate these organisms failed to detect the illness-inciting strains or that the virulence of these strains had declined following initial isolation. Additionally, we must consider the possibility that the embryo lethality assay of virulence used here might not be sensitive enough to detect differences between these two groups of isolates. Also, it should be noted that virulence assays, such as the one used here, fail to account for predisposing host or environmental conditions, enabling a less virulent isolate to cause disease under natural conditions. Interestingly, the complement resistance of a strain was significantly associated with its lethality in embryos, and iss-containing isolates were significantly more likely than those lacking iss to be classified as complement-resistant and virulent. Such results, at least for this group of avian E. coli, suggest that there is a compelling but imperfect relationship among complement resistance, virulence, and the presence of iss. These results also suggest that the flow cytometric assay may be a reasonable alternative to the viable count method of determining complement resistance.     

Virulence of South African isolates of Haemophilus paragallinarum. Part 2: naturally occurring NAD-independent field isolates

Naturally occurring NAD-independent variants of Haemophilus paragallinarum, which have been isolates from poultry showing clinical signs of infectious coryza, were used to determine their virulence using a newly developed challenge model for infectious coryza. It was established that the NAD-independent isolates belonging to a particular serogroup, were less virulent when compared to the virulence of the NAD-dependent isolates from the same serogroup. It was shown that the virulence of the NAD-independent isolates belonging to serogroup C and serogroup A were very similar to each other. This differs to the results obtained with NAD-dependent isolates reported on previously, in which the serogroup C isolates were found to be more virulent then the serogroup A isolates.     

Comparative pathogenicity of three genetically distinct Trypanosoma congolense-types in inbred Balb/c mice

Inbred Balb/c mice were infected with three clones of Trypanosoma congolense (Sam.28.1, Dind.3.1 and K60.1A) corresponding, respectively, to the three genetically distinct types (savannah, forest and kilifi) defined within this species, for the purpose of comparing their pathogenicity for a better understanding of the epidemiology of African trypanosomosis. Another clone of savannah type, IL 3000, was also tested simultaneously to study a probable strain variation. Both the clones of savannah type were found of extreme virulence with loss of appetite, rough hair, rapid respiration, lethargy, and all mice died within a week. Parasitaemias evolved rapidly to the first peak by day 3-5 post-inoculation without any remission and the course of disease was correlated positively with the prepatent period. The clones of the forest type and the kilifi type were of low virulence with chronic infection and symptoms progressively less patent throughout the infection; only one mouse died in each experimental group.     

Virulence of recent isolates of Mycoplasma synoviae in turkeys

Systemic Mycoplasma synoviae (MS) infection was induced experimentally in commercial turkeys with recent MS isolates (K4822D and K4774J) from turkey breeder flocks that exhibited no clinical signs typical of MS infection except for a low incidence of swollen footpads. The virulence of each strain was compared by evaluating gross and microscopic lesions, serologic responses, and MS isolation rates at 10 and 21 days postchallenge and by comparing these results with those obtained from a known virulent isolate (K1968), another previously characterized field isolate (K4463B), and unchallenged controls. All strains induced lesions typical of infectious synovitis but showed distinct differences in the extent of the gross and microscopic lesions and in the isolation rates from the tissues in turkeys. K1968 induced the most extensive lesions in hock and stifle joints and footpads, but strains K4822D, K4774J, and K4463B all induced synovitis and were similar in virulence for synovial tissues. Very mild respiratory lesions were induced by all of the strains studied. All strains yielded strong positive serologic responses. We concluded that these recent field isolates, although able to induce synovitis, are less virulent for turkeys than a known virulent strain. Nevertheless, under severe experimental challenge, these strains have the capability of causing lesions that may be incompatible with economical turkey production.     

Susceptibility of N'Dama cattle to experimental challenge and cross-species superchallenges with bloodstream forms of Trypanosoma congolense and T. vivax.

Susceptibility to Trypanosoma congolense, T. vivax challenge and cross species-superchallenges, and related effects on health and productivity were assessed in N'Dama cattle. Twenty-five N'Dama bulls aged 3-4 years and previously primed with trypanosome infections through natural tsetse exposure over more than one year were used. The experimental herd was divided in five groups each composed of five randomly selected animals. Group 1 was challenged with T. congolense, Group 2 with T. vivax, Group 3 was inoculated with T. congolense followed by a cross-superchallenge with T. vivax, Group 4 was inoculated with T. vivax followed by T. congolense cross-superchallenge. Animals in Group 5 were used as controls. Both T. vivax and T. congolense cross-superchallenges were carried out on Day 14 subsequent to respective initial T. congolense and T. vivax inoculations. All challenges were performed by intradermal needle inoculation of stocks of trypanosome bloodstream forms. In challenged animals (Group 1 to 4), parasitaemia profiles and packed red cell volumes (PCV) were measured for four months. Weight changes were recorded monthly and daily weight gain (DWG) computed. All cattle challenged with T. congolense became parasitaemic. Conversely, one animal in Group 2 and two in Group 3 never displayed patent T. vivax parasitaemia. Both in single (Group 1), initial (Group 3) and cross-superchallenged (Group 4) cattle higher percentage of positive blood samples and higher parasitaemia level were obtained following T. congolense than T. vivax inocula (Group 2, 3 and 4) (P<0.04 or greater). Overall the pre-challenge period, PCV values and DWGs were nearly identical in the five groups. Conversely, over the post-challenge period, cattle singly, initially and cross-superinoculated with T. congolense (Group 1, 3 and 4) displayed lower PCV values and DWGs in comparison with both control animals (Group 5) and with singly T. vivax challenged cattle (Group 2) (P<0.05 or greater). No difference in mean PCV levels and DWGs was found between animals in Group 2 and cattle in Group 5. It was concluded that trypanotolerant N'Dama cattle suffered more from T. congolense and mixed T. congolensel T. vivax infections, while pure T. vivax infection did not produce appreciable negative effects on their health and productivity. Therefore, considering that tsetse and trypanosomosis control campaigns are costly and are justified only when derived economic benefits exceed those of control, and also that an ample mosaic of farming systems exists in West Africa, species-specific trypanosome prevalence and relative impact should be assessed in various cattle populations and breeds differing in trypanosome susceptibility before advising any intervention. Moreover, virulence and related effects of T. congolense and T. vivax endemic stocks on health and productivity in local cattle populations should also be estimated in order to counsel appropriate economic protection measures against trypanosmosis, i.e. vector control and/or strategic use of trypanocidal drugs.     

Effect of strains of mice and challenge dose on the infectivity and virulence of Trypanosoma vivax

Factors influencing the infectivity and virulence of clone-derived Trypanosoma vivax for mice were investigated using a titration technique capable of recognizing differences in magnitude of about 10-fold. Marked differences were observed in the susceptibility and duration of infection in four inbred strains of mice after low dose (10(2.2)ID63) challenge. Only mild differences in survival period were observed when challenge doses of 10(3.2) and 10(4.2)ID63 per mouse were used. In all tests, Balb/C mice were most susceptible while CBA mice were most resistant. Long survival of infected mice was highly correlated with frequent occurrence of remission of parasitaemia. Infectivity of T. vivax isolates varied during the course of an infection, being high at the early stages of the infection and very low at later stages.     

Virulence of recent and former classical swine fever virus isolates evaluated by their clinical and pathological signs

The clinical diagnosis of classical swine fever (CSF) still caused problems to the veterinarians during the last decade. The primary CSF outbreak was often detected too late and, meanwhile, the virus had spread. Consequently, the recent classical swine fever virus isolates (CSFV) were suspected to be of low virulence. The purpose of the study was to quantify the virulence of four recent CSFV by evaluating the clinical and pathological signs caused by different CSFV. Pigs of the same breed and age group were inoculated intranasally with CSFV from recent epidemics in European Union (EU) member states. The CSFV used are registered in the data base of the EU Reference Laboratory for CSF and belong to different genotypes: 2.1, 2.2 and 2.3 respectively. Clinical signs of CSF were evaluated by using a score system suggested previously (Mittelholzer et al., 2000: Vet. Microbiol. 74, 293). For the evaluation of pathological lesions, a new pathological score was introduced. The four CSFV tested here were classified as moderately virulent in general, although one CSFV may cause different clinical courses, ranging from highly virulent to avirulent. This indicates the importance of additional factors in the host animal for virulence. Differences in the clinical and pathological signs between these four recent CSFV were rather minor, emphasizing that the genetic typing of CSFV is absolutely essential. Differences towards former CSFV (e.g. reference virus strain Alfort 187) were more pronounced, especially regarding the onset and duration of the disease, the occurrence of skin haemorrhages and pathological lesions of kidney, subcutis and serosae. It is concluded that clinical diagnosis of CSF is rather difficult in pigs up to 14 days post-CSFV infection using these four CSFV, emphasizing the need for careful differential diagnosis and the laboratory investigation for CSF at an early stage.     

Serum resistance as an indicator of virulence of Pasteurella multocida for turkeys

Wildlife isolates of Pasteurella multocida, whose virulence for turkeys had previously been determined by intravenous inoculation, were characterized regarding their ability to survive incubation in fresh non-immune turkey serum. The relative virulence of the isolates was significantly associated with their ability to resist the bactericidal power of the serum as determined by standard plate counts following incubation. Organisms with a high survival value were more virulent; those with a low survival value were less virulent. A statistical model was specified and was successfully used to predict relative virulence of the P. multocida isolates. This method of assaying serum resistance was rapid, repeatable, and practical and could be performed with minimal laboratory equipment. Also studied was the serum resistance of seven serotype 3, 4 isolates obtained from the lungs of M9-vaccinated turkeys from seven flocks experiencing increased mortality due to fowl cholera. These isolates were shown to be identical to the M9 vaccine by restriction endonuclease analysis of chromosomal DNA. Six of the seven isolates had higher serum survival values than the original M9 vaccine.     

High tissue burden of Toxoplasma gondii is the hallmark of acute virulence in mice

Toxoplasma gondii virulence is commonly determined by mortality rate of infected mice. Limited data showed that virulent T. gondii strains had increased parasite growth in mice compared to that of less virulent strains. To determine if this is a common phenomenon for a variety of strains and to develop an alternative assay to test acute virulence in mice, we measured parasite burdens in experimentally infected outbred CD-1 mice for 19 T. gondii isolates, in which the virulence phenotypes had previously been determined by mortality assay. Our results showed that parasite concentrations in spleen tissues were two orders of magnitude higher in the virulent than the intermediately and non-virulent isolates at day 7 post infection. In competition assays, mice inoculated with mixed tachyzoites of virulent and intermediately virulent strains or virulent and non-virulent strains showed that the former always reached a higher concentration at day 7 post infection. In mixed infection of intermediate and non-virulent strains, both strains were detectable in mice at day 7 post infection. In conclusion, our data showed that the virulence of T. gondii can be predicted by parasite load in the spleen tissue of infected mice at 7 days post infection, providing an alternative method to determine virulence of Toxoplasma.