European Swine Research Articles

Lack of cross-protection between European H1N1 and H1N2 swine influenza viruses

Background: Swine influenza viruses (SIVs) of H1N2 subtype have recently become established in several European countries, and they cocirculate with H1N1 and H3N2 viruses. The H1N2 virus haemagglutinin (HA) appears to be of human origin and fails to cross-react with avian-like H1N1 SIVs in vitro. This study examines whether in vivo cross-protection occurs between H1N1 and H1N2 viruses isolated in Belgium. Methods: Influenza virus-seronegative pigs were inoculated first with Sw/Gent/7625/99 (H1N2) or Sw/Belgium/1/98 (H1N1), or left uninoculated. Four weeks later, all pigs were challenged with the H1N2 virus. We examined H1N2 antibody titres prior to challenge, and clinical signs and virus replication after challenge. Results: H1N2 antibodies were found exclusively in the pigs previously infected with H1N2, and these were protected against disease and infection. Fever and respiratory signs typical of H1N2 infection developed in the challenge control pigs and the H1N1-immune pigs. In both groups, all pigs had H1N2 virus in the lungs at 24 h and in nasal swabs during the first week after challenge. In H1N1-immune pigs, however, the mean virus titre in the lungs was 1.9 log 10 EID 50/g lower than in the challenge controls. Similarly, the total amount of virus excreted was significantly reduced and virus excretion was on the average 1.4 days shorter. Conclusions: Pigs immune after H1N1 subtype infection are not protected against H1N2 infection and disease. This is in agreement with the antigenic difference in the HAs of these viruses. Our data, however, suggest partial heterosubtypic immunity between H1N1 and H1N2, which may be mediated by the cytotoxic T lymphocyte response.

Quantitative TaqMan® RT-PCR for the detection and differentiation of European and North American strains of porcine reproductive and respiratory syndrome virus

Since two different types of porcine reproductive and respiratory syndrome virus (PRRSV), the European (EU) and the North American (US) strain, occur or coexist in European swine herds, their rapid and reliable detection and differentiation is essential for disease surveillance. A quantitative TaqMan® reverse transcription-polymerase chain reaction (RT-PCR) is described for PRRSV detection and strain differentiation. Sensitivity and specificity were compared with a conventional PRRSV RT-PCR and to the detection of both PRRSV types in cell cultures and both were found to be equal or superior to the reference methods. Reproducibility was tested and proved that the assay was very reliable. Standard dilutions included in each test allowed absolute quantitation of the amount of viral RNA. The TaqMan® assay described below is time-saving, easy to handle, exhibits a decreased risk of cross-contamination and is highly sensitive and specific. It is, therefore, considered to be a powerful tool for the rapid detection and differentiation of PRRSV.

Infection of a child in Hong Kong by an influenza A H3N2 virus closely related to viruses circulating in European pigs.

Influenza virus A/Hong Kong/1774/99, isolated from a young child with mild influenza, was shown to be similar in its antigenic and genetic characteristics to H3N2 viruses circulating in pigs in Europe during the 1990s and in particular to be closely related to viruses isolated from two children in the Netherlands in 1993. Similar viruses had previously not been identified outside Europe. Although there is little evidence as to how the child contracted the infection, it appears likely that pigs in southern China were the source of infection. Characteristics shared with the European swine viruses include resistance to the anti-influenza drugs amantadine and rimantadine. Thus not only does this incident once again highlight the potential of pigs as a source of novel human influenza viruses, but also indicates the potential for emergence of amantadine-resistant human viruses.

Antigenic and Genetic Characterization of Swine Influenza A (H1N1) Viruses Isolated from Pneumonia Patients in The Netherlands

It is generally believed that pigs can serve as an intermediate host for the transmission of avian influenza viruses to humans or as mixing vessels for the generation of avian–human reassortant viruses. Here we describe the antigenic and genetic characterization of two influenza A (H1N1) viruses, which were isolated in The Netherlands from two patients who suffered from pneumonia. Both viruses proved to be antigenically and genetically similar to avian-like swine influenza A (H1N1) viruses which currently circulate in European pigs. It is concluded that European swine H1N1 viruses can infect humans directly, causing serious disease without the need for any reassortment event.

Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals.

Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3'SL-PAA and 6'SLN-PAA, which contained, respectively, 3'-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6'-sialyl(N-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6'SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q-->L, increased binding to 6'SLN-PAA, while among H1 swine viruses, the 190E-->D and 225G-->E mutations in the HA appeared important for the increased affinity of the viruses for 6'SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.

Independence of evolutionary and mutational rates after transmission of avian influenza viruses to swine.

In 1979, an H1N1 avian influenza virus crossed the species barrier, establishing a new lineage in European swine. Because there is no direct or serologic evidence of previous H1N1 strains in these pigs, these isolates provide a model for studying early evolution of influenza viruses. The evolutionary rates of both the coding and noncoding changes of the H1N1 swine strains are higher than those of human and classic swine influenza A viruses. In addition, early H1N1 swine isolates show a marked plaque heterogeneity that consistently appears after a few passages. The presence of a mutator mutation was postulated (C. Scholtissek, S. Ludwig, and W. M. Fitch, Arch. Virol. 131:237-250, 1993) to account for these observations and the successful establishment of an avian H1N1 strain in swine. To address this question, we calculated the mutation rates of A/Mallard/New York/6750/78 (H2N2) and A/Swine/Germany/2/81 (H1N1) by using the frequency of amantadine-resistant mutants. To account for the inherent variability of estimated mutation rates, we used a probabilistic model for the statistical analysis. The resulting estimated mutation rates of the two strains were not significantly different. Therefore, an increased mutation rate due to the presence of a mutator mutation is unlikely to have led to the successful introduction of avian H1N1 viruses in European swine.

Continued Evolution of H1N1 and H3N2 Influenza Viruses in Pigs in Italy

Swine influenza viruses possessing avian genes were first detected in Europe in 1979 (Scholtisseket al.,1983,Virology,129, 521–523) and continue to circulate in pigs in that region of the world. To characterize the molecular epidemiology of swine influenza viruses currently circulating in Europe, we used dot-blot hybridization and sequence analysis to determine the origin of the genes encoding the nonsurface proteins (“internal” genes) of 10 H1N1 and 11 H3N2 swine influenza viruses isolated in Italy between 1992 and 1995. All of the 126 genes examined were of avian origin; thus the currently circulating H3N2 strains which possess A/Port Chalmers/1/73-like surface glycoproteins appear to be descendants of the reassortant human–avian viruses that emerged between 1983 and 1985 in Italy. Sequence analysis of matrix (M), nonstructural, and nucleoprotein genes, as well as phylogenetic analysis of M gene showed that the H1N1 and H3N2 viruses from the pigs were closely related to recent isolates of the avian-like swine H1N1 influenza strain currently circulating in northern Europe and were distinguishable from the genes of viruses isolated from European swine in 1979. To evaluate the frequency of transmission of swine H1N1 and H3N2 viruses to man, we tested 123 human sera for hemagglutination-inhibiting antibodies against avian and mammalian H1N1 and H3N2 virus strains. Our findings indicate that swine influenza viruses possessing A/Port Chalmers/1/73-like hemagglutinin may have transmitted to approximately 20% of young persons under 20 years of age who had contact with pigs. Thus, H3N2 swine viruses, possibly possessing avian-derived internal genes, may be entering humans more often than was previously thought. We strongly recommend that pigs be regularly monitored as a potential early warning system for detection of future pandemic strains.

Use of nonradioactive cDNA probes to differentiate porcine respiratory coronavirus and transmissible gastroenteritis virus isolates.

Porcine respiratory coronavirus (PRCV), a member of the family Coronaviridae, is antigenically related to transmissible gastroenteritis virus (TGEV) of swine. PRCV, now thought to be a mutant of TGEV, was first isolated in 1984 from pigs in Belgium that were seropositive for TGEV but did not have a history of clinical transmissible gastroenteritis (TGE). Since the initial isolation of PRCV, it has been found that infections of swine in Europe with PRCV are widespread. PRCV has also been isolated in swine in the United States, 7,11,20,24 but its prevalence in herds within the United States is not known. There are several similarities between PRCV and TGEV. Both viruses have 3 major structural proteins: the surface spike (S) glycoprotein, the integral membrane glycoprotein, and an internal nucleoprotein. Nucleotide sequences of PRCV isolates thus far studied show that they are closely related to TGEV but that there are some striking differences. PRCV isolates have a characteristic deletion in the 5' end of the S gene when compared to TGEV, and PRCV has a different tissue tropism than TGEV. TGEV replicates in both the respiratory and intestinal tissues and causes gastroenteritis, 14 whereas PRCV replicates to high titers in lung tissue of swine and with little or no replication in the intestinal tissues and no evidence of gastroenteritis and villous atrophy. PRCV is antigenically related to TGEV in that polyclonal sera which neutralize TGEV also neutralize PRCV. Thus, conventional serologic methods are not useful in determining if a swine herd with anti-TGEV antibodies has been infected with PRCV or TGEV. Anti-TGEV neutralizing monoclonal antibodies (MAbs) directed against the S glycoprotein readily neutralize PRCV; however, there are some nonneutralizing anti-TGEV MAbs directed against the S glycoprotein that can be used to distinguish between PRCV and TGEV isolates in a competitive binding assay. Of the European PRCV isolates that have had their nucleotide sequences published, all have a 672-nucleotide deletion in the 5' end of the S gene. The US PRCV isolates Ind/89 and ISU-1 have a 681-nucleotide deletion present in the 5' end of the S gene. Recently the PRCV isolates AR310 and LEPP have been shown to have a smaller S gene deletion of 621 nucleotides present. Additionally, the PRCV isolate IA1894 has recently been shown to have a 678-nucleotide deletion in the 5' end of its S gene. Hence, a cDNA probe that encompasses the region of the TGEV S gene that is characteristically deleted from PRCV isolates can be used

Interspecies transmission of influenza viruses.

In this report we examine the hypothesis that aquatic birds are the primordial source of all influenza viruses in other species. Two partly overlapping reservoirs of influenza A viruses exist in migrating water-fowl and shorebirds throughout the world. These species harbor influenza viruses of all the known hemagglutinin and neuraminidase subtypes. In contrast to the rapid, progressive changes in both the nucleotide and amino acid sequences of mammalian virus gene lineages, avian virus genes show far less variation and, in most cases, appear to be in evolutionary stasis. There are periodic exchanges of influenza virus genes or whole viruses between species, giving rise to pandemics of disease in humans, lower animals, and birds. The periodic exchange of influenza viruses between species has been illustrated by the appearance of new pandemic influenza viruses in humans, including the Spanish influenza of 1918, the Asian influenza of 1957, and the Hong Kong influenza of 1968. Transmission of avian influenza viruses to swine in Europe in 1979 has resulted in the appearance of human-avian reassortant influenza viruses in pigs in Italy and in children in the Netherlands. These studies provide evidence supporting the possibility that pigs serve as a mixing vessel for reassortment between influenza viruses in mammalian and avian hosts and raise the question of whether the avian influenza viruses now circulating in European swine are the precursors of the next human pandemic virus.

European Swine Virus as a Possible Source for the Next Influenza Pandemic?

According to phylogenetic data, about 100 years ago an avian influenza virus passed the species barrier (possibly first) to pigs and (possibly from there) to humans. In 1979 an avian influenza A virus (as a whole, without reassortment) again entered the pig population in northern Europe, forming a stable lineage. Here it is shown that the early North European swine viruses exhibit higher than normal evolutionary rates and are highly variable with respect to plaque morphology and neutralizability by monoclonal antibodies. Our results are consistent with the idea that, in order to pass the species barrier, an influenza A virus needs a mutator mutation to provide an additional number of variants, from which the new host might select the best fitting ones. A mutator mutation could be of advantage under such stress conditions and might enable a virus to pass the species barrier as a whole even twice, as it seems to have happened about 100 years ago. This stressful situation should be over for the recent swine lineage, since the viruses seem to be adapted already to the new host in that the most recent isolates — at least in northern Germany — are genetically stable and seem to have lost the putative mutator mutation again.

Molecular evolution of influenza viruses.

There are two different mechanisms by which influenza viruses might evolve: (1) Because the RNA genome of influenza viruses is segmented, new strains can suddenly be produced by reassortment, as happens, for example, during antigenic shift, creating new pandemic strains. (2) New viruses evolve relatively slowly by stepwise mutation and selection, for example, during antigenic or genetic drift. Influenza A viruses were found in various vertebrate species, where they form reservoirs that do not easily mix. While human influenza A viruses do not spread in birds and vice versa, the species barrier to pigs is relatively low, so that pigs might function as "mixing vessels" for the creation of new pandemic reassortants in Southeast Asia, where the probability is greatest for double infection of pigs by human and avian influenza viruses. Phylogenetic studies revealed that about 100 years ago, an avian influenza A virus had crossed the species barrier, presumably first to pigs, and from there to humans, forming the new stable human and classical swine lineages. In 1979, again, an avian virus showed up in the North European swine population, forming another stable swine lineage. The North European swine isolates from 1979 until about 1985 were genetically extremely unstable. A hypothesis is put forward stating that a mutator mutation is necessary to enable influenza virus to cross the species barrier by providing the new host with sufficient variants from which it can select the best fitting ones. As long as the mutator mutation is still present, such a virus should be able to cross the species barrier a second time, as happened about 100 years ago. Although the most recent swine isolates from northern Germany are again genetically stable, we nevertheless should be on the lookout to see if a North European swine virus shows up in the human population in the near future.

Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) are encoded by the carboxyterminal portion of viral open reading frame 3

Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were revealed by serologic analysis of a recombinant protein derived from PRRSV open reading frame 3 (ORF 3). The hydrophilic carboxyterminal 199 amino acids encoded by the ORF 3 of a European (Lelystad) isolate of PRRSV were expressed as a recombinant fusion protein (BP03-P) in a baculovirus gene expression system. Sera from gnotobiotic swine exposed to prototypic reference European and American isolates of PRRSV and sera from conventionally reared European and American swine convalescing from naturally acquired PRRSV infections were used to characterize the BP03-P protein. Sera from gnotobiotic and conventionally reared swine exposed to European isolates of PRRSV were significantly more reactive ( P < 0.01) with BP03-P than were the corresponding American PRRSV antisera using the indirect immunoperoxidase monolayer assay (IPMA). Prototypic European, but not American, PRRSV antisera also recognized BP03-P using western immunoblotting and radioimmunoprecipitation assay (RIPA) procedures. However, gnotobiotically derived antiserum to an atypical American-origin PRRSV was reactive with BP03-P by both IPMA and western immunoblot. Despite a predicted potential for N-linked glycosylation, studies with tunicamycin and peptide-N-glycosidase F (PNGase F) indicated that BP03-P was not N-glycosylated in either insect cell cultures or Trichoplusia ni larvae infected with the recombinant baculovirus. Sera from rabbits inoculated with BP03-P failed to neutralize both the European (Lelystad) and American (ATCC VR-2332) reference isolates of PRRSV and did not react by IPMA with PRRSV-infected cell cultures. Taken together, the data suggest that the carboxyterminal portion of PRRSV ORF 3 encodes a non-neutralizing viral peptide that is partially responsible for the serologic differences noted between European and most American isolates of PRRSV.

Infection of Children with Avian-Human Reassortant Influenza Virus from Pigs in Europe

Pigs have been proposed to act as the intermediate hosts in the generation of pandemic human influenza strains by reassortment of genes from avian and human influenza virus strains. The circulation of avian-like H1N1 influenza viruses in European pigs since 1979 and the detection of human-avian reassortants in pigs raises the question of whether these viruses actually have the potential to transmit and cause disease in humans. We now report the serologic and genetic characterization of two human influenza A viruses (A/Netherlands/5/93 [H3N2] and A/Netherlands/35/93 [H3N2]) that caused influenza in children in The Netherlands in 1993. The results show that these viruses are human-avian reassortants that were generated and currently still are circulating in European swine. This shows the pivotal role that pigs can play in the generation and transmission of avian influenza virus genes to humans and their potential to generate a new human pandemic strain.

Genetic relatedness between influenza A (H1N1) viruses isolated from humans and pigs

Complete nucleotide sequences were obtained from the nucleoprotein genes of three influenza A viruses and partial nucleotide sequences were obtained from the polymerase, neuraminidase, matrix, and non-structural protein genes of four influenza A viruses that had been isolated between 1931 and 1939 from clinically sick pigs in the United States or Europe. A phylogenetic analysis of the open reading frames of nine nucleoprotein genes showed that the U.S. swine influenza virus isolates from 1931 and 1937 originated from the classic swine viral nucleoprotein lineage, whereas the European swine strains fro 1938 and 1939 were placed among the early human influenza virus nucleoprotein lineage. All the partial gene sequences obtained from the two European swine strains fro 1938 and 1939 were also more closely related to early human H1N1 reference strains than to the U.S. swine isolates from 1931 and 1937, indicating that none of the four viruses isolated from swine had acquired genes from a heterologous lineage through reassortment.

Estrogen associated changes in uterine blood flow and maternal recognition of pregnancy in Chinese (Meishan) pigs

Our laboratory has recently demonstrated a reduced embryonic growth rate and estradiol-17β (E 2β) content of preimplantation Meishan embryos compared with embryos of European breeds. Estrogen secretion by the preimplantation conceptus has been linked to both the transient increase in uterine blood flow (UBF), and maternal recognition of pregnancy (MRP) on Days 11–13 of gestation in European swine breeds. The objective of these experiments was to evaluate the temporal association between estrogen secretion by the preimplantation conceptus, increases in UBF and the onset of MRP in Meishan pigs. In Experiment 1, multiparous Meishan sows ( N = 3) were observed for estrus once daily, and hand-mated at first observed estrus (Day 0) and 24 h later with a Meishan boar. On Day 2 postmating, sows were bilaterally instrumented with blood flow transducers and uterine venous catheters. UBF (ml min −1) was monitored and blood samples collected once daily from Days 6 to 30 postmating. UBF increased on Day 11 ( P = 0.06) to peak on Day 13, then decreased on Days 14–17, before increasing progressively thereafter, coincident with increasing uterine E 2β secretion. In Experiment 2, second cycle Meishan ( N = 20) and Yorkshire ( N = 18) gilts were hand-mated at first observed estrus (Day 0), and 24 h later, to boars of their own breed. Embryos were flushed from the gilts on Day 9, 11, 13 or 15 postmating. Gilts were again observed for estrus to determine treatment estrous cycle length (ECL). Treatment ECL was similar ( P > 0.1) to presurgical ECL when embryos were collected from either Meishan or Yorkshire gilts on Days 9 and 11. ECL was extended in both breeds ( P < 0.05) when embryos were recovered on Days 13 and 15.

A sero-epizootiological study of porcine respiratory coronavirus in Belgian swine.

A porcine respiratory coronavirus (PRCV), antigenically closely related to transmissible gastroenteritis virus (TGEV), appeared in the European swine population in 1984. The present serological study was performed to obtain insight into the epizootiology of PRCV and of TGEV. PRCV-induced neutralizing antibodies were found in 90.6 per cent of the 160 sera collected from sows at slaughter, demonstrating the enzootic appearance of PRCV in the Belgian swine population. A serological study of fattening swine on 33 farms revealed that 11 farms situated in an area with a high farm density (all farms within 4 km2) and 11 on 22 closed breeding-fattening farms situated in areas with a low farm density (only one to four farms per 12 km2) were infected with PRCV throughout the year, whereas the other 11 closed breeding-fattening farms were temporarily free of PRCV. PRCV disappeared from the farms mainly in spring and summer. All the 11 farms became reinfected in autumn or winter, indicating that PRCV is regularly reintroduced in farms in the colder seasons. There was no correlation between the herd size and the temporary disappearance of PRCV from farms. It was observed on some farms that PRCV could infect pigs shortly after weaning in the presence of declining maternal antibodies, indicating that PRCV can persist on a farm by regularly infecting newly weaned pigs. TGEV-specific antibodies were found in 7.6 per cent of the 160 sera from the slaughterhouse sows. TGEV-specific antibodies were also detected in sera from fattening swine of 5 of the above mentioned 33 farms. TGEV-outbreaks were not observed on these farms.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide sequence analysis of the HA 1 coding portion of the haemagglutinin gene of swine H1N1 influenza viruses

The nucleotide and deduced amino acid sequences coding for the HA 1 portion of the haemagglutinin (HA) genes of three swine influenza viruses were determined and compared with published HA sequence data for human H1N1 influenza viruses. Sequence differences between the classic swine influenza HAs sw37 (A/swine/29/37) and NJ76 (A/New Jersey/11/76) were randomly distributed in the molecule without being confined to antigenic sites. In contrast, sequence differences between the HAs of sw37 and the antigenically atypical strains sw38 (A/swine/Northern Ireland/38) and sw39 (A/swine/Cambridge/39) were clustered in hypervariable regions, similar to the pattern of changes that was present between sw37 and the human strains PR834 (A/PR/8/34) and WSN33 (A/WSN/33). Sequence homologies of the European swine influenza strains (sw38, sw39) were higher with the HAs of the human strains (PR834, WSN33) than with the classic swine influenza HAs (sw37, NJ76). Phylogenetic analysis showed that the HA genes of these two European swine influenza strains emerged from a different evolutionary lineage of H1 HAs than the HAs of classic swine influenza strains.

Pathogenesis of classical swine fever: B-lymphocyte deficiency caused by hog cholera virus

Hog cholera, also known as classical or European swine fever, is caused by hog cholera virus, a member of the genus Pestivirus. It is shown here that the end stage of lethal infection in the natural host is associated with a dramatic depletion preferentially of B lymphocytes in the circulatory system as well as in lymphoid tissues. Already at the onset of disease, viral replication in lymphoid tissues demarcates the germinal centers, and the viral genome remains localized to that site as the disease progresses even after morphologic disintegration of the follicular structure. A block in B-lymphocyte maturation by infection and destruction of germinal centers is discussed as a key event in the pathogenesis of acute, lethal hog cholera.

Split Guard Hairs in Both Domestic and European Wild Swine

Split Guard Hairs in Both Domestic and European Wild Swine

African swine fever virus.

The causative agent of African swine fever (ASF) is an icosahedral virus 175 to 215 mv, in diameter. It is sensitive to lipid solvents and contains deoxyribonucleic acid (DNA). Thus far, it appears to have no close relatives among other viruses that infect mammals. It has apparently existed for a very long time in wild swine indigenous to Africa, for with them it has established an ideal host-parasite relationship in which prolonged infection occurs without signs of disease. In fact, the existence of the virus came to light only after domestic swine of European origin were brought into areas inhabited by wart hogs. Although the precise mode of transmission from wild swine is obscure, the virus spreads readily among domestic swine and produces a peracute disease with mortality approaching 100 per cent. Since the swine industry is quite sparse in such areas, the outbreaks are largely self-limiting. Each outbreak usually represents a new incursion of the virus from its natural reservoir. This continues to be the pattern of most outbreaks in Africa, and until recently the peracute infection which prevails under these conditions has been regarded as the usual form of the disease in domestic swine. However, ASF has spread to Europe and in some areas has become enzootic in domestic swine. In these areas, acute, subacute, and chronic infections are common and the mortality is somewhat reduced. Its resemblance to classical swine fever (American hog cholera and European swine fever) has become more pronounced, and differential diagnosis in the field is seldom possible.