Correlation of serum concentration of alpha 1-acid glycoprotein with lymphocyte blastogenesis and development of experimentally induced or naturally acquired hepatic abscesses in cattle.

Changes in serum alpha 1-acid glycoprotein (alpha 1AG) concentration in cattle with hepatic abscesses were observed, and function of alpha 1AG was evaluated, particularly its influence on cellular immune response. Test cattle (n = 4) were inoculated with Fusobacterium necrophorum, control cattle (n = 2) were inoculated with inactivated bacteria, and naturally affected cattle (n = 11) were found in a slaughterhouse. Determination of alpha 1AG was made by use of a single radial immunodiffusion method. The action on lymphocyte blastogenesis was determined by [3H]thymidine incorporation. Cultured lymphocytes from healthy cattle were treated with variable concentrations of alpha 1AG purified from serum obtained from cattle with hepatic abscesses and suppression of blastogenesis stimulated by each of 3 mitogens was measured. In cattle with experimentally induced abscesses, serum alpha 1AG concentration increased for 7 to 10 days after F necrophorum inoculation, its change being parallel to that of sialic acid. High concentration of alpha 1AG was found in naturally affected cattle and was highly correlated to sialic acid concentration. Suppression of lymphocyte blastogenesis in cattle with experimentally induced hepatic abscesses was highly correlated to serum alpha 1-AG concentration.     

The pathogenesis of persistent turbinate atrophy induced by toxigenic Pasteurella multocida in pigs

Six one-week-old piglets were pretreated with a 1% acetic acid solution for two days in one or both nostrils. Three piglets were not treated with acetic acid. Three days after treatment all nine piglets were inoculated in both nostrils with a toxigenic type D strain of Pasteurella multocida. Three piglets were killed seven days after inoculation; one died spontaneously 13 days after inoculation and the remaining pigs were killed at approximately 90 kg body weight, i.e., five to six months of age. All acetic acid-treated animals developed severe atrophy of the turbinates in the treated nostrils. Untreated nostrils were normal. The present results showed that toxigenic P. multocida can induce turbinate atrophy that persisted until 90 kg body weight when the lesions were similar to spontaneous atrophic rhinitis in pigs. The turbinate atrophy was not accompanied by inflammatory reaction, atrophy of other bone structures, or lesions in other organs. The experiment showed furthermore that toxigenic P. multocida may be present in the tonsils of control animals without causing turbinate atrophy. A pathogenesis for atrophic rhinitis in pigs is proposed.     

Experimental Babesia Divergens Infection in Reindeer (Rangifer Tarandusj

Two non-splenectomized reindeer developed fever, icterus and haemoglobinuria after inoculation intravenously with cattle blood containing Babesia divergens.14 and 15 days after inoculation agglutination of Babesia-containing erythrocytes from the two reindeer was observed in blood films. In one reindeer there was also microscopical evidence of intravascular agglutination in the sinusoids of the spleen.As a control, a non-splenectomized reindeer calf was inoculated with normal cattle blood; there were no signs of agglutination.     

Infection with Ostertagia ostertagi in goats and calves

In order to determine the usefulness of the goat as a model host for Ostertagia ostertagi, a series of experiments was conducted in which young goats and calves were experimentally infected with L3 of calf-source and goat-source isolates. The goat-source isolate was derived from a continuous passage of the bovine parasite in goats. Patent infections resulted in 73 out of 86 inoculated goats (85%). The largest number of patent infections was observed when inoculation consisted of a single dose of goat-source larvae. Percent establishment of infection was generally low in goats inoculated with either larval source. Time taken to achieve patency in goats was frequently within the range normal for cattle infections, but was often extended (21-67 days). With the exception of the generally higher level of establishment of goat- or calf-source isolates in calves and the low frequency of the vulval flap in adult female worms established in goats, little difference was observed in percent establishment or worm population characteristics of the two isolates in goats as based on source of larval inoculum, inoculation course, and age of host at inoculation. Prolonged passage of infection in goats did not result in stabilized isolate more adapted to the goat or less adapted to calves. Fecal egg counts were generally minimal or negative in goats during the first 30 days of infection, but were often increased and not substantially lower than counts in calf infections after 60 or 90 days. Low level egg counts in goats were observed to persist for up to 17 months. During the spring of 2 years, goat kids grazed on a cattle pasture acquired O. ostertagi infections which included adult worms, but a larger number of early L4. The latter were presumed to be inhibited in development just as such inhibition occurs in cattle during spring.     

Humoral and cellular immune responses in cattle and sheep inoculated with Sarcocystis

Cattle inoculated with Sarcocystis bovicanis (= Sarcocystis cruzi) and sheep inoculated with Sarcocystis ovicanis were monitored for the appearance of Sarcocystis-specific antibodies and lymphocytes in the peripheral circulation. Anti-Sarcocystis antibody was identified by enzyme-linked immunosorbent assay, whereas antigen-reactive lymphocytes were discerned by an in vitro lymphocyte blastogenic assay. The antigens used were the soluble fraction recovered from disrupted bradyzoites of mature sarcocysts. Cattle developed anti-Sarcocystis immunoglobulin (Ig)M responses, beginning 3 to 4 weeks after inoculation, and IgG1 antibody responses, beginning 5 to 6 weeks after inoculation. The increase in IgM antibody was relatively brief, returning to near preinfection levels in 2 to 3 months. In contrast, IgG1 antibody levels remained high for at least 5 to 6 months. Neither IgG2 nor IgA antibody responses were demonstrable in cattle. In sheep, the IgG antibody levels followed a time course similar to that seen in cattle, except that the increase was slightly delayed (6 to 8 weeks after inoculation was done). Measurable IgM antibody response was not seen in sheep. Cellular immunoresponsiveness as judged by in vitro lymphocyte blastogenesis in cattle was different from that in sheep. Sarcocystis-specific lymphocytes were demonstrable in the circulation of cattle within 15 days after they were inoculated, but the activity decreased rapidly. In sheep, reactive cells were not evident until 3 to 4 weeks after inoculation were done, but peripheral blood lymphocytes taken from these sheep as long as 5 to 6 weeks after the inoculations remained capable of mounting strong blastogenic responses. Neither the enzyme-linked immunosorbent assay nor the blastogenic assay showed species specificity. Animals immunized with a given species of Sarcocystis gave similar in vitro responses to antigens from the immunizing species and to other species of Sarcocystis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of an ELISA for the diagnosis of experimentally induced and naturally occurring Leptospira hardjo infections in cattle

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Leptospira interrogans serovar hardjo (hardjo) infection in cattle was compared with the microscopic agglutination test (MAT). Glutardialdehyde was used in the ELISA to couple sonicated hardjo antigen to the microtiter plate. Mouse monoclonal anti-bovine IgG1 coupled to peroxidase was used as conjugate. Sera from calves experimentally inoculated with hardjo reacted positively in the MAT as early as 10 days after inoculation; these sera did not react positively in the ELISA until 25 days after the first inoculation. Positive and negative field sera from 704 adult cattle on 90 farms were examined by the MAT and the ELISA; a 90% correlation between the two tests was demonstrated. Eighty-six sera from calves inoculated with four Leptospira serogroups other than hardjo and 227 field sera from adult cattle with naturally occurring leptospirosis other than hardjo were examined by the ELISA. Fewer than 1% of these heterologous sera reacted with hardjo antigen in the ELISA. We concluded that the ELISA described in this report is an advantageous alternative to the MAT for diagnosing leptospirosis.     

Development of a neutralising antibody response to an inoculated cytopathic strain of bovine virus diarrhoea virus

Fourteen clinically healthy cattle that were persistently infected with non-cytopathic bovine virus diarrhoea virus (BVDV) and three BVDV-free cattle were inoculated with one of three cytopathic BVDV strains. Mucosal disease developed in 12 of the viraemic cattle, resulting in a moribund condition 17 to 99 days after inoculation. Two of the viraemic cattle remained clinically healthy until the end of the experiment, 14 months after inoculation. The BVDV-free cattle did not develop clinical signs after inoculation. From each cow with mucosal disease a noncytopathic and a cytopathic BVDV strain were isolated from tissue specimens collected post mortem. All the cattle developed moderate to high levels of neutralising antibodies against the cytopathic BVDV strain with which they were inoculated. The antibodies from 10 of the 12 cattle with mucosal disease did not react with the cytopathic BVDV strains isolated post mortem, and antibodies from none of them reacted with the non-cytopathic BVDV isolates. Antibody responses to the inoculated BVDV strains developed earlier in the viraemic cattle than in the BVDV-free cattle.     

Development and serial passage of persistent lymphocytosis associated with bovine leukemia virus infection in cattle

Two calves each were inoculated with 1.5 x 10(8) or 5 x 10(9) lymphocytes collected from each one cow which had persistent lymphocytosis (PL) and antibodies to bovine leukemia virus (BLV). A sudden increase in the number of peripheral blood lymphocytes (PBL) was observed 14 and 23 days, respectively, after inoculation and the maximum number reached 29,000 and 52,000/microliters 72 and 57 days after inoculation. Although the degree of PL decreased gradually in these cattle, it continued until 14 and 44 months after inoculation when one animal was sacrificed and the other died of lymphosarcoma. The PL was passaged in cattle by inoculation of a large number of PBL obtained from cattle at the stage of PL (PLL). The degree of PL was severer in cattle inoculated with a larger number of PLL. PL was not caused by inoculation of PBL obtained from either BLV-infected non-PL cattle or cattle free of BLV. The PL was also caused by inoculation of PLL into BLV-infected non-PL cattle. On the other hand, it was not observed after inoculation of a large amount of cell-free virus obtained from short-term cultures of PLL. Antibodies to BLV developed earlier and to higher levels in cattle inoculated with PLL than in those inoculated with cell-free virus. These facts show that infection with BLV was established more effectively by PLL than by cell-free virus, the infection may occur by lymphocyte to lymphocyte interaction and the actual number of infected BLV may have an important role in development of PL.     

Role of the spleen in the pathogenesis of experimentally induced bovine leukemia virus infection

In an extension of a previous pathogenesis study, bone marrow and other tissues from four experimentally inoculated cattle were tested for virus between the 13th and 20th days after experimental inoculation with bovine leukemia virus. BLV was detected in the blood of three, spleen of two, lymph node of two and bone marrow of only one of the inoculated cattle. In additional studies, four splenectomized and two intact control calves were also examined. Two of these calves were splenectomized before BLV inoculation and two after a persistent virus infection had been established. Results indicated that the removal of the spleen affected neither the establishment and persistence of virus infection nor the development and maintenance of serological responses to viral antigens.     

Multiplication of attenuated and virulent porcine parvoviruses in colostrum-deprived, neonatal pigs

Colostrum-deprived, neonatal, 2 days old pigs were inoculated with the attenuated HT-/SK or the virulent 90HS strain of porcine parvovirus (PPV) by the oral or subcutaneous route and sacrificed 2, 4 or 6 days after inoculation. Then, comparison was made on viral multiplication in pigs between the two strains. Pigs inoculated with the HT-/SK strain showed no detectable viremia or HI antibody responses against PPV within 6 days after inoculation. Only in pigs inoculated by the subcutaneous route, a small amount of virus was recovered from the spleen, liver, or mesenteric lymph nodes. These viruses were distinguished from the parental virulent 90HS strain, as examined for rct maker in vitro. When pigs were inoculated with the virulent 90HS strain, viremia appeared in all of them 1 day after inoculation and continued for up to the sacrificed day. Moreover, a considerable amount of virus was also detected from all tissues, including brain, lung, liver, spleen, pancreas, small intestine, and lymph node tissues, in all pigs tested. HI antibodies were first detected 6 days after inoculation.     

TUBERCULIN SENSITIVITY OF CATTLE INOCULATED WITH ATYPICAL MYCOBACTERIA ISOLATED FROM CATTLE, FERAL PIGS AND TROUGH WATER

Each of 12 cattle was inoculated either subcutaneously and intradermally or into a mesenteric lymph node with 1 of 8 species of live atypical mycobacteria isolated from cattle, cattle trough water and feral pigs. Seventy-eight days after inoculation the cattle were tuberculin tested with bovine PPD, avian PPD and homologous heat-concentrated syntheic medium tuberculins. They were killed 85 days after inoculation. Organisms were cultured from caseous granu-lomas at all sites in cattle inoculated with M. avium serotype 2. M. simiae was recovered from a granuloma at the subcutaneous site. Acid-fast bacilli were isolated from the mesenteric lymph node inoculated with trough water organisms. At 72 h, all the cattle had produced skin reactions of 4 mm or more to the homologous tuberculins and all except 1 produced a similar response to avian PPD. Only isolates of bovine origin sensitised cattle to bovine PPD to this degree, and these reactions were less than the corresponding response to avian PPD.     

水貂肠炎细小病毒灭活疫苗抗体消长规律的研究

应用血凝抑制试验对水貂肠炎细小病毒灭活疫苗免疫水貂进行抗体水平动态监测,结果表明,疫苗接种14 d抗体达到保护,21~30 d抗体水平达到高峰;免疫180 d抗体仍在保护值以上。攻毒试验证实,免疫180 d后90%免疫水貂获得保护,确定水貂细小病毒性肠炎灭活疫苗免疫保护期可持续6个月。     

Transmission of classical swine fever virus by artificial insemination.

Classical swine fever (CSF) virus was introduced into an artificial insemination centre during the CSF epizootic of 1997-1998 in the Netherlands. The risk of further spread of CSF virus via contaminated semen was recognised, but could not be assessed because scientific data on this issue were not available. An animal experiment was performed to determine whether CSF virus could be transmitted via artificial insemination with contaminated semen. Three boars were inoculated with a CSF virus field isolate and from Day 5 till Day 18 thereafter, ejaculates were collected and prepared for insemination. Ruttish sows were inseminated with the extended semen from Day 5 till Day 18 after inoculation of the boars. All the inoculated boars remained healthy throughout the experiment and developed CSF neutralising antibodies between 14 and 21 days after inoculation. Virus was isolated from several semen samples collected from 5 till 11 days after inoculation. Two out of six sows inseminated with CSF contaminated semen seroconverted after insemination. All the other sows remained seronegative. In the foetuses of both the seropositive sows, CSF virus was detected at approximately 35 days post insemination. These results demonstrate that adult boars infected with CSF virus can excrete virus with semen and can, subsequently, transmit the virus to sows and their foetuses via artificial insemination.     

Immunity to Hammondia hammondi infection in cats.

Acquisition of immunity to Hammondia hammondi, a newly recognized coccidian of cats, was studied in 18 specific-pathogen-free cats. One cat was given a single oral inoculation, 11 cats were given 2 oral inoculations, and 1 cat was given 3 oral inoculations of homogenized mouse carcasses containing H hammondi. In all cats, oocyst shedding began 6 to 9 days after the 1st inoculation. Oocyst shedding peaked at 1 to 2 days after the onset of shedding and lasted for 1 to 2 weeks. None of the cats became sick. Of the 11 cats inoculated twice (between 2-51 days after the 1st inoculation), 5 shed oocysts 7 to 14 days after the repeat inoculation; however, fewer oocysts were shed at this time. One cat that was inoculated thrice (14 and 51 days after the 1st inoculation) shed oocysts 14 to 17 days after the 3rd inoculation but not after the 2nd inoculation. Spontaneous oocyst shedding was studied in 9 of these 13 H hammondi-infected cats for 5 months. Two cats spontaneously shed oocysts: One cat (inoculated only once) spontaneously re-shed oocysts 21 to 24, 31 to 33, 49 to 50, and 118 to 120 days after inoculation; The other cat (inoculated twice-the 2nd time, 6 days after the 1st inoculation) re-shed oocysts 38 to 48, 85 to 89, and 133 to 136 days after the 1st inoculation. The course of H hammondi infection was studied in 5 cats given weekly injections of 6-methyl prednisolone acetate for at least 7 weeks, starting 18 days before inoculation in 2 cats, and starting 14, 34, and 45 day after inoculation in 3 cats. The induced hyperadrenocorticism did not affect the prepatent period or induce parasitism of extraintestinal organs. The 3 cats infected for 14, 34, and 45 days, re-shed oocysts after hyperadrenocorticism was induced. It was concluded that immunity to H hammondi infection in cats is less stable than immunity to the related coccidian, Toxoplasma gondii.     

Lesion development in white-tailed deer (Odocoileus virginianus) experimentally infected with Mycobacterium bovis

The recent discovery of tuberculosis in free-living white-tailed deer in northeastern Michigan underscores the need for increased understanding of the pathogenesis of tuberculosis in wildlife species. To investigate lesion development in white-tailed deer, 32 deer were experimentally infected by intratonsilar instillation of 300 colony-forming units of Mycobacterium bovis. Three deer each were euthanatized and examined at days 15, 28, 42, and 56 after inoculation, and five deer each were euthanatized and examined at days 89, 180, 262, and 328 after inoculation. Microscopic lesions first were seen in the medial retropharyngeal lymph node and lung 28 and 42 days after inoculation, respectively. Lung lesions were present in 12 (38%) of 32 deer, involving 23 lung lobes. Left caudal and right middle and caudal lobes were involved in 17 (74%) of the 23 affected lung lobes. Lesions in the medial retropharyngeal lymph node first appeared as granulomas composed of aggregates of macrophages and Langhans-type giant cells. Some early granulomas contained centrally located neutrophils. As granulomas developed, neutrophils were replaced with a central zone of caseous necrosis that first showed signs of mineralization 42 days after inoculation. Granulomas increased in size as the zone of caseous necrosis expanded. Peripheral fibrosis, first seen at 56 days after inoculation, progressed to only a thin fibrous capsule by 328 days after inoculation. By the termination of the study, the central necrotic core of the granuloma contained abundant liquefied necrotic material and grossly resembled an abscess. Although tuberculous lesions in white-tailed deer follow a developmental pattern similar to that in cattle, fibrosis is less pronounced and the advanced lesions may liquefy, a change seldom reported in cattle. An understanding of lesion development will aid in the identification of the spectrum of disease that may be seen in this important wildlife reservoir of tuberculosis.     

Infection of African buffalo (Syncerus caffer) and cattle with Theileria parva lawrencei after serial passage in cattle

The infectivity of a Theileria parva lawrencei stabilate, from a stock derived from an African buffalo (Syncerus caffer) in the Serengeti National Park, Tanzania, was investigated. In the first experiment a buffalo and three cattle were inoculated with a stabilate from a stock passaged three times in cattle. All cattle developed fatal theilerial infections. Isolations from the buffalo by tick feeding and cell culture isolation showed that it was infected with T p lawrencei at the time of inoculation, but the second isolation made 19 days after inoculation behaved like T p parva in cattle, developing a high parasitosis, while the third isolation made three months later behaved like T p lawrencei with low parasitosis. It was concluded that two biological types of T parva could exist in a buffalo at one time, but it was not shown that the buffalo had become a carrier of T p lawrencei adapted to cattle. In the second experiment two buffaloes and three cattle were inoculated with T p lawrencei (Serengeti) stabilate which had been passaged six times through cattle and ticks. The two buffaloes had mild theilerial infections and developed serological titres in the indirect fluorescent antibody test, but the cattle had fatal infections. Tick and cell culture isolations of T parva were possible during the clinical reactions of the buffaloes, but no carrier state was demonstrated. Theileria-infected cell lines were established from the buffaloes and the cattle and were examined using monoclonal antibodies against T parva schizonts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inoculation of conventionally and specific-pathogen-free reared rabbits with Ostertagia ostertagi isolated from cattle.

Eleven trials were conducted to collect helminthologic and pathologic data from 27 conventionally reared (CR) and 53 specific-pathogen-free (SPF) 6- to 13-week-old male New Zealand White rabbits. These rabbits were given 50,000 to 100,000 ensheathed third-stage infective Ostertagia ostertagi larvae (L3) orally. The L3 had been isolated from the feces of cattle. Fecal egg counts were conducted and worm populations were determined after euthanasia 3 to 56 days after inoculation. At necropsy, nodules were observed in a confluent pattern in the mucosa of cardiac region of the stomach in all inoculated rabbits, except in CR rabbits inoculated at 6 weeks of age, which had no nodules or worm burdens 42 days after inoculation. Confluent areas were not observed in SPF rabbits euthanatized 5 days or less after inoculation; however, small, transparent nodules were evident in the mucosa of the cardiac region of the stomach. Fourth-stage larvae (L4) were obtained from the mucosal nodules after digestion of stomach of both types of rabbits. There were more L4 in SPF than CR rabbits. In addition, greater numbers of L4 were found in SPF rabbits euthanatized 14 days or less after inoculation. Petechial hemorrhages were in the fundic area of the stomach mucosa in SPF rabbits inoculated with 100,000 L3 and euthanatized 14 days later. Mature O ostertagi or worm eggs in feces were not found in inoculated CR or SPF rabbits. The pathologic changes had characteristics similar to those in cattle and goats infected with O ostertagi.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental transmission of chronic wasting disease agent from mule deer to cattle by the intracerebral route.

This communication reports final observations on experimental transmission of chronic wasting disease (CWD) from mule deer to cattle by the intracerebral route. Thirteen calves were inoculated intracerebrally with brain suspension from mule deer naturally affected with CWD. Three other calves were kept as uninoculated controls. The experiment was terminated 6 years after inoculation. During that time, abnormal prion protein (PrP(res)) was demonstrated in the central nervous system (CNS) of 5 cattle by both immunohistochemistry and Western blot. However, microscopic lesions suggestive of spongiform encephalopathy (SE) in the brains of these PrP(res)-positive animals were subtle in 3 cases and absent in 2 cases. Analysis of the gene encoding bovine PRNP revealed homozygosity for alleles encoding 6 octapeptide repeats, serine (S) at codon 46, and S at codon 146 in all samples. Findings of this study show that although PrP(res) amplification occurred after direct inoculation into the brain, none of the affected animals had classic histopathologic lesions of SE. Furthermore, only 38% of the inoculated cattle demonstrated amplification of PrP(res). Although intracerebral inoculation is an unnatural route of exposure, this experiment shows that CWD transmission in cattle could have long incubation periods (up to 5 years). This finding suggests that oral exposure of cattle to CWD agent, a more natural potential route of exposure, would require not only a much larger dose of inoculum but also may not result in amplification of PrP(res) within CNS tissues during the normal lifespan of cattle.     

Experimental inoculation of cattle with bovine herpesvirus-4: evidence for a lymphoid-associated persistent infection

A strain of bovine herpesvirus-4 isolated from cows with mammary pustular dermatitis was used for experimental inoculation of cattle. This strain is serologically indistinguishable from the group prototype Movar 33/63 and from strain DN-599. Seronegative cattle were inoculated IV or by simultaneous intranasal, IV, intramammary (via teat channel), and intradermal inoculations. All inoculated cattle seroconverted. Clinical signs of disease or lesions were not evident, except for a dermal lesion corresponding with one intradermal inoculation site. Virus was recovered from the dermal lesion and was excreted in the milk for 17 days. Virus was recovered from esophagopharyngeal fluid at 9 and 13 days after inoculation. At different times of euthanasia (2 to 14 months after inoculation), virus was recovered from cocultures with bovine lung cells and/or explant cultures of lymph nodes, spleen, tonsils, and, in one case, kidney. In 2 animals, the virus was recovered repeatedly during 1 year from peripheral blood leukocytes by cocultivation with bovine lung cells. The number of infectious leukocytes, as determined by infectious center assay, ranged from less than 1 to 6 infectious cells/10(7) leukocytes.     

Transmission and virological studies of a malignant catarrhal fever syndrome in the Indonesian swamp buffalo (Bubalus bubalis)

A malignant catarrhal fever-like syndrome in indonesian swamp buffalo was experimentally transmitted to one of 2 Bos indicus and 3 of 3 Bos javanicus cattle by intravenous inoculation of 250 ml of citrated, whole blood from affected buffaloes. The 4 cattle developed clinical signs of disease on average 32.5 days after receiving the inoculation of blood. The 4 cattle died after a variable period of illness. None of a further 3 B. javanicus cattle inoculated intravenously with a spleen homogenate prepared from another affected buffalo developed the disease. The experimental disease was clinically and pathologically similar to the natural disease in buffaloes although differences were noted. Attempts to adapt the agent to mice, guinea pigs and rabbits failed. A cytopathic agent (Japanese encephalitis virus) was isolated from the spleen of one buffalo with clinical signs but was not considered significant. Sixty-three B. indicus, 7 B. javanicus (and 6 of their crosses), 3 B. taurus and 4 Bubalus bubalis (Murrah buffalo) were kept in the same quarters where 50 of 177 swamp buffaloes died between September 1979 and May 1982. Four of the 7 B. javanicus cattle developed the clinical signs of disease and died. All the other cattle in contact remained healthy.