Scanning electron microscopy of digestive tract parasites in jungle cat (Felis chaus).

Among the 10 native cats of Iran, the Felis chaus is considered as one of the largely unknown species. Despite the reports on jungle cats in southeast Iran, there is no comprehensive information about their parasitic diseases. Scanning electron microscope (SEM) provides a valuable tool for the study of these organisms. In October 2022, a 15-year-old male jungle cat (F. chaus) was examined for gastrointestinal parasites using SEM which revealed exact parasite structures such as the mouth, shape of the lips, teeth and their position, papillae, excretory pore, and amphids. SEM of the Taenia taeniaeformis found in the F. chaus intestinal epithelium revealed that this tapeworm consisted of four unarmed suckers and a large armed rostellum. The rostellum was observed bearing double rings of alternating and circular hook arrangements. There were 21 large hooks in the anterior ring and 21 slightly smaller hooks in the posterior one. This study is the first report of Joyeuxiella echinorhyncoides from a jungle cat (F. chaus) in southeastern Iran. The rostellum was cylindrical and armed with thorn shape hooks that covered the whole rostellum with similar density. It was armed with 20-30 long hooks arranged in 16 rows. Four big suckers were visible at the base of the rostellum, two on each side. The suckers were round, and unlike the rostellum, these structures were unarmed. The results of the present study observed under SEM, indicated that organs such as sucker and rostellum in cestodes and mouth and the shape of lips and teeth in nematodes played a significant role in the biology of these helminths. The SEM reveals the surface structures and their relationships which results in better distinguishing the species and showing their detailed differences. More SEM studies need to be conducted on other species of cestodes and nematodes, along with pathological tests, to obtain more comprehensive and precise information about how these parasites harm this jungle cat host. The results of present study indicate that morphological details are important which should be considered in the classification. Meanwhile, external structures described herein using SEM, further investigation using transmission electron microscope provide more data on the biology of the parasites. RESEARCH HIGHLIGHTS: Light microscopy is the first step to identify the parasites. High magnification 3D images of the SEM reveal the surface structures of parasites and their relationships precisely. SEM study of the parasites results in distinguishing the species and showing their detailed differences.

Cardio-pulmonary parasites of the European wildcat (Felis silvestris) in Germany.

BACKGROUND: In the last years, research on feline cardio-pulmonary parasites has considerably increased in Europe. Not only domestic cats (Felis catus), but also European wildcats (Felis silvestris) can serve as definitive hosts for these nematodes. The F. silvestris population in Germany has been growing rapidly within the last decades; therefore, the assessment of its cardio-pulmonary parasite status is of importance to unravel whether the wildcat population serves as a substantial reservoir for these nematodes and might pose a health threat to domestic cats. METHODS: As part of a nature conservation project for European wildcats in the German federal state Rhineland-Palatinate, lungs (n = 128) and hearts (n = 111) of 128 F. silvestris found dead were examined for cardio-pulmonary parasites. All isolated parasites were identified morphologically, and results were confirmed by molecular analysis of a total of 3-11 specimens of each worm species. RESULTS: A total of 70.3% (90/128) wildcats were positive for at least one lungworm species. Angiostrongylus chabaudi was most common (53.1% [68/128]), followed by Aelurostrongylus abstrusus (42.2% [54/128]), Troglostrongylus brevior (31.3% [40/128]) and Capillaria spp. (3.1% [4/128]). Of note, about two-thirds of the infected wildcats harboured coinfections. Infection intensities ranged from 1 to 167 nematodes per wildcat. Generalised linear models revealed a strong correlation between A. chabaudi and A. abstrusus infection, and prevalences were higher in adult than in younger wildcats, except for T. brevior. Moreover, the T. brevior prevalence varied significantly with nutritional status. CONCLUSIONS: This study shows that feline cardio-pulmonary nematodes are common parasites in European wildcats in Germany but do not appear to have a serious impact on the overall health of the population. Due to presumed spillover events via prey, cardio-pulmonary nematodes may circulate between the wildcat population and domestic cats and might therefore pose a health risk to individual domestic cats.

First description of peritoneal and pleural metacestodosis caused by Mesocestoides vogae in a European wild cat (Felis silvestris silvestris).

Tapeworms of the genus Mesocestoides (Cestoda: Cyclophyllidea: Mesocestoididae) are still enigmatic to scientists, due to their high morphological variability, low host specificity, and unknown details of their life cycle. They are found worldwide, with carnivorous mammals as the main definitive hosts, and the disease is potentially zoonotic. After ingestion by a definitive host, the tetrathyridium can occasionally migrate through the intestinal wall and reach the peritoneal cavity or abdominal organs causing peritoneal metacestodosis. Here, we report on a case of metacestodosis of a European wild cat (Felis silvestris silvestris) found dead in Croatia. At necropsy, a large number of white, rice-like structures were found free in the abdominal and thoracic cavities, as well as along the serous surfaces and in the lungs. DNA isolated from the nodules was genotyped and based on a 320-base pair long 12S fragment classified as Mesocestoides vogae. Although post-mortem changes were advanced, severe emaciation due to the severe parasitic infection and gastrointestinal bleeding was diagnosed as the likely cause of death. Intestinal cestodosis was previously reported in wild cats, but according to our knowledge, this is the first description of peritoneal and pleural metacestodosis caused by M. vogae tetrathyridia (metacestodes) in any wild carnivore species.

Molecular screening indicates high prevalence and mixed infections of Hepatozoon parasites in wild felines from South Africa.

Genetic diversity within partial 18S rRNA sequences from Hepatozoon protozoan parasites from wild felines in South Africa was assessed and compared with data from domestic cats to assess patterns of host specificity. Lions, leopards, servals, a caracal and an African wildcat were all positive for parasites of the Hepatozoon felis-complex. However, haplotypes were not species-specific, and potential mixed infections were widespread. Additional genetic markers are needed to untangle the extremely complex situation of these parasites in both domestic cats and wild felines in South Africa.

Severe Verminous Pneumonia Caused by Natural Mixed Infection with Aelurostrongylus abstrusus and Angiostrongylus chabaudi in a European Wildcat from Western Balkan Area.

AIMS: Aelurostrongylus abstrusus (Railliet, 1898) and Angiostrongylus chabaudi (Biocca, 1957) are important cardiopulmonary metastrongyloids in felids. This case report describes, for the first time, a natural and patent mixed infection caused by A. abstrusus and A. chabaudi in a European wildcat (Felis silvestris silvestris) from Bosnia and Herzegovina. Most important, this is a rare report of fatal parasitism in wildlife, involving a severe verminous bronchopneumonia and gastrointestinal nematode and cestode infection with Toxocara cati, Taenia taeniaeformis, Aonchotheca putorii, and Ancylostoma spp. RESULTS: Emphasis is set to detailed description of granulomatous, interstitial verminous bronchopneumonia, morphological description of A. abstrusus and A. chabaudi, and molecular confirmation of diagnosis by triplex PCR. CONCLUSION: The data provided in this study contribute to the knowledge on the epizootiology and pathological effect of these neglected metastrongyloids in European wildcat.

Use of a commercial serologic test for Angiostrongylus vasorum for the detection of A. chabaudi in wildcats and A. daskalovi in badgers.

Three species of the genus Angiostrongylus are known to infect European carnivores: A. vasorum (mainly in canids but also in other carnivores), A. chabaudi (in felids) and A. daskalovi (in mustelids). A. vasorum is responsible for clinically severe disease in domestic dogs, most commonly diagnosed based on fecal examination and serological detection of circulating antigens. Considering the poorly known host range and the challenging larval differentiation in the feces between the three species of Angiostrongylus infecting European carnivores, our aim was to evaluate the cross-reactivity of A. chabaudi and A. daskalovi with A. vasorum using a commercial serologic test developed for domestic dogs. Badgers (Meles meles) (n=10) and wildcats (Felis silvestris) (n=8) were examined between 2015 and 2016 by full parasitological necropsy with subsequent morphological and molecular identification of nematodes and by serology, using IDEXX Angio Detect™ tests. Five out of the ten badgers and two out of the eight wildcats were harboring nematodes in the pulmonary arteries. All nematodes were identified morphologically as A. daskalovi in badgers and A. chabaudi in wildcats, respectively. Serological examination of the plasma samples revealed the positivity of the same animals as found in necropsy. None of the animals negative at necropsy was positive at serology. The 100% correlation between the necropsy results and the serologic positivity to IDEXX Angio Detect™ in badgers infected with A. daskalovi and wildcats infected with A. chabaudi suggest that these rapid tests are able to identify circulating antigens of all species of Angiostrongylus found in European carnivores: A. vasorum, A. daskalovi and A. chabaudi. The possibility for future in-clinic use of this test in domestic cats should be further investigated.

Spirometra erinaceieuropaei in a wildcat (Felis silvestris) in Iran.

The zoonotic helminth, Spirometra, has several species with almost global distribution. Herein, we describe the first detailed molecular detection of Spirometra erinaceieuropaei in a road-killed wildcat (Felis silvestris) in Iran and its identification at the species level using CO1 gene. Genomic DNA was extracted using CTAB extraction method. The DNA then was applied for PCR amplification of cytochrome c oxidase subunit I (CO1) gene. Afterwards, PCR product was sequenced and obtained data were analyzed and multiple aligned using BLAST program, ClustalX and Bioedit software. Microscopy findings and diagnostic clues revealed that the parasite is a Spirometra sp. cestode. Consequently, molecular analysis on the basis of cytochrome c oxidase subunit 1 (CO1) gene demonstrated that the species is Spirometra erinaceieuropaei. Regarding optimum climate conditions and previous reports of animal infection in this region, the likelihood of human involvement should be potentially considered.

First detection of Cytauxzoon spp. infection in European wildcats (Felis silvestris silvestris) of Italy.

Cytauxzoonosis is an emerging, tick-transmitted, protozoan disease affecting domestic and wild felids and caused by Cytauxzoon felis, Cytauxzoon manul and Cytauxzoon spp. This study aimed to determine the presence of infection with Cytauxzoon spp. in Felis silvestris silvestris in Italy, in order to enhance the comprehension of its pattern distribution among domestic cat populations. In addition, wildcats were tested for other endemic vector-borne pathogens in Italy. The carcasses of 21 F. s. silvestris were collected from central and northern regions of Italy. All the animals were submitted to necropsy and samples of the spleens were collected. Cytauxzoon infection was surveyed by a conventional PCR amplifying a portion of the SSU-rDNA of species of Piroplasmida. The samples were also screened for Anaplasma spp., Ehrlichia spp., Rickettsia spp., Babesia spp., Theileria spp., and Leishmania spp. using SYBR Green Real-Time PCR (rPCR) assays. Four animals (19%) were positive for Piroplasmida-PCR assay and three sequenced amplicons were obtained (14.3%), clustering with the Italian, Spanish, French and Romanian Cytauxzoon spp. isolates and with C. manul found in Mongolia. The samples were negative for the other pathogens screened. The present results showed that Cytauxzoon spp. may infect both F. s. silvestris and F. s. catus.

Angiostrongylus chabaudi (Biocca, 1957) in wildcat (Felis silvestris silvestris, S) from Romania.

Angiostrongylus chabaudi is a rare cardio-pulmonary nematode infecting felids. Although almost 60 years have passed since the original description of the species in Italy, this parasite has been seldom found in domestic and wildcats in southern Europe. The present study aims to report a new case of patent A. chabaudi infection in a road-killed wildcat from Maramureș County in Northern Romania. The necropsy revealed the presence of parasites in the pulmonary arteries and the right ventricle, and the fecal examination showed the presence of L1 larvae. Parasites were morphologically and morphometrically characterized as A. chabaudi, showing 100 % nucleotide similarity to an Angiostrongylus sp. originating from a wildcat from Germany and 99 % to A. chabaudi from Italy. This study reports A. chabaudi for the first time in Eastern Europe, expanding knowledge about the distribution range of this species.

OCCURRENCE OF LUNGWORMS IN EUROPEAN WILDCATS (FELIS SILVESTRIS SILVESTRIS) OF CENTRAL ITALY.

The increasing focus on infections in domestic cats ( Felis catus ) has raised questions about lungworm distribution in wild hosts. To enhance knowledge of the occurrence of lungworms in enzootic regions of central Italy, we examined the carcasses of 16 European wildcats ( Felis silvestris silvestris). Adult nematodes, feces, respiratory flushings, and pulmonary tissues were collected at necropsy and then microscopically and genetically analyzed. Fourteen wildcats had single or mixed lungworm species. Aelurostrongylus abstrusus was the most common parasite retrieved, followed by Troglostrongylus brevior. In addition, three specimens of Angiostrongylus chabaudi were found in the pulmonary arteries of one wildcat. Histologically, the most common lesions were a mild-to-severe chronic catarrhal bronchitis and a chronic interstitial pneumonia with smooth muscle hypertrophy, associated with T. brevior and A. abstrusus , respectively. These results demonstrate that the European wildcats may harbor several species of lungworms that may impair their health and welfare. Also, F. s. silvestris is a potential reservoir for respiratory nematodes in domestic cats.

Cytauxzoon Infections in Wild Felids from Carpathian-Danubian-Pontic Space: Further Evidence for a Different Cytauxzoon Species in European Felids.

Parasitic protists of the genus Cytauxzoon are detected in a wide range of wild and domestic felids. Bobcats are a confirmed reservoir of Cytauxzoon felis in North America while domestic cats are susceptible hosts suffering from severe or fatal illness. Cytauxzoon infections are mainly reported from American felids and, recently, several sub-clinical and clinical findings were reported from European, Asian, and African felids. In 2014, the collection of organs of 4 Eurasian lynx and 12 wild cats from 11 Romanian localities was carried out to determine the prevalence and genetic diversity of Cytauxzoon spp. We detected an overall high prevalence of 62.5% in both species of wild felids; 50% in wild cats and 100% in Eurasian lynx. The phylogenetic analysis indicates 2 distinct clades of Cytauxzoon in felids, with all of our sequences clustering with sequences of Cytauxzoon sp./Cytauxzoon manul from Palaearctic felids. Further studies, development of new genetic markers, and experimental transmission studies are required for clarifying the taxonomy and life cycle of feline Cytauxzoon in the Old World.

First evidence of the European wildcat (Felis silvestris silvestris) as definitive host of Angiostrongylus chabaudi.

Angiostrongylus chabaudi (Strongylida, Angiostrongylidae) is a parasitic nematode described for the first time last century from the pulmonary arteries of six European wildcats (Felis silvestris silvestris) in central Italy. Since then, this parasite remained practically unknown until recently, when immature A. chabaudi have been reported from one wildcat in Germany and two domestic cats (Felis silvestris catus) in Italy. The present report describes the first record of A. chabaudi in Greece and, most importantly, the first known case of patent infection by A. chabaudi. The necropsy of a road-killed F. s. silvestris found near the lake Kerkini, in the municipality of Serres (Macedonia, Greece), revealed the presence of nematodes of both sexes in the right ventricle and the pulmonary artery of the heart. All parasites were mature adults and numerous eggs were present in the uteruses of females. The morphological characteristics of the parasites were consistent with those of A. chabaudi. Moreover, Angiostrongylus-like first stage larvae (L1) were present in the faeces of the animal that was negative for any other cardio-pulmonary parasite. Genetic examination of adult parasites and L1 confirmed the morphological identification as A. chabaudi. Histopathological examination of the lungs showed severe, multifocal to coalescing, chronic, interstitial granulomatous pneumonia due to the presence of adult parasites, larvae and eggs. These findings demonstrate for the first unequivocal time that this nematode reproduces in the European wildcat which should be ultimately considered a definitive host of A. chabaudi. Finally, the L1 of A. chabaudi are described here for the first time, opening new prospects for further studies on this neglected parasite.

Survey on parasitic infections in wildcat (Felis silvestris silvestris Schreber, 1777) by scat collection.

Wildcats are endangered felid species living in Europe, Asia, and Africa. Regrettably, scientific information on parasites of wildcats is particularly meager and they often rely on data gained by necropsies of a small number of animals. In the present study, scat collection was used to assess the parasite spectrum of European wildcats living in the Etna Park (Sicily, Italy). Scat collection was performed from May to September 2010 by weekly walking four transects for a total of 391 km. Samples were then analyzed by flotation and sedimentation techniques to investigate wildcat parasitic fauna. A total of 121 scats of wildcats were collected, and parasitic forms (i.e., oocysts, eggs, and larvae) were retrieved in 110 (90.9 %) of the samples. Parasites found were Physaloptera sp. (52.1 %), tapeworms (45.5 %), Toxocara cati (43.8 %), Eucoleus aerophilus (27.3 %), Ancylostoma sp. (22.3 %), Troglostrongylus brevior (15.7 %), trematodes (9.9 %), Isospora felis (4.1 %), Cylicospirura sp. (1.7 %), and Acanthocephala (0.8 %). The prevalence of endoparasitic infections herein recorded is similar to that described in other studies conducted using necropsy technique. The species richness of parasites found in the present survey, with a total of nine helminths and one protozoon, is the highest ever reported for wildcat in Europe. Scat collection and examination are reliable and rapid non-invasive tools which can be used in a systematic survey design to study the parasite spectrum of wildcat as well as that of other endangered wild species.

Genetic characterisation of Toxoplasma gondii isolates from European beavers (Castor fiber) and European wildcats (Felis silvestris silvestris).

Six free-ranging European beavers (Castor fiber) from Berlin greater metropolitan area and twelve European wildcats (Felis silvestris silvestris) originating from the German Federal State of Saxony-Anhalt were found dead and their carcasses were submitted for necropsy. Brain and lung samples were analysed for the presence of Toxoplasma gondii DNA. Histo-pathologic analysis of one beaver revealed several cyst-like protozoal structures in parts of the brain. Tissue DNA isolated from all animal samples was analysed by a specific T. gondii-PCR. Two beavers and four wildcats tested T. gondii-positive. DNA of the parasites was further analysed by PCR-RFLP typing using nine markers (nSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico). Only T. gondii type II alleles were detected, except for the Apico locus, where type I alleles were observed in two isolates from beavers and in three from wild cats. The results of this study indicate that type II T. gondii (including type II variant strain) is the most common genotype infecting wildcats and beavers from Germany.

Toxoplasmosis in Sand cats (Felis margarita) and other animals in the Breeding Centre for Endangered Arabian Wildlife in the United Arab Emirates and Al Wabra Wildlife Preservation, the State of Qatar.

The Sand cat (Felis margarita) is a small-sized felid found in sand and stone deserts ranging from the north of Africa to Asia, with the Arabian Peninsula as its centre of distribution. The Sand cat captive breeding program at the Breeding Centre for Endangered Arabian Wildlife (BCEAW), Sharjah, UAE, has experienced high newborn mortality rates, and congenital toxoplasmosis was recently recognized as one of the causes of this mortality. In the present study, one 18-month-old Sand cat (FM019) died of acute toxoplasmosis-associated hepatitis and pneumonitis acquired after birth; Toxoplasma gondii was demonstrated in histological sections which reacted with T. gondii polyclonal antibodies by immunohistochemistry (IHC). T. gondii DNA was found by PCR of extracted DNA from liver and lung tissues of this cat. Antibodies to T. gondii were found in serum examined in 1:1600 dilution in the modified agglutination test (MAT); its 2-year-old cage mate seroconverted (MAT titer 1:3200) at the same time. Another Sand cat (FM017) was euthanized because of ill health when 3 years old; its MAT titer was >1:3200, and T. gondii tissue cysts were found in brain, heart, ocular muscles and skeletal muscle, confirmed by IHC. Viable T. gondii was isolated by bioassays in mice inoculated with tissues of another chronically infected Sand cat (FM002); T. gondii was not found in histological sections of this cat. T. gondii antibodies were found in several species of animals tested, notably in 49 of 57 wild felids at BCEAW. A 7-year-old Sand cat (3657) from Al Wabra Wildlife Preservation (AWWP), Doha, State of Qatar died of acute visceral toxoplasmosis with demonstrable T. gondii tachyzoites by IHC, and T. gondii DNA by PCR, and a MAT titer of >3200. T. gondii antibodies were found in 21 of 27 of wild felids at AWWP. PCR-RFLP genotyping at 10 genetic loci revealed that these T. gondii isolates from Sand cat (FM002 and FM019) at BCEAW have an atypical genotype, which was previously reported in T. gondii isolates of dogs from Sri Lanka. The genotype from the cat from AWWP (3657) is a genetic Type II strain with a Type I allele at locus Apico. This is the first report of genetic characterization of T. gondii isolates from Middle East.

Seroprevalence of antibodies to Toxoplasma gondii in Gordon's wildcat (Felis silvestris gordoni) in the Middle East.

Most species of felids tested have been found to be the definitive host for Toxoplasma gondii. Gordon's wildcat (Felis silvestris gordoni) is a threatened species found in the United Arab Emirates (UAE) and Oman. Antibodies to T. gondii were found in all 29 captive and 2 of 7 wild-caught F. s. gordoni in UAE examined by the modified agglutination test (MAT). Titers were 1:100 in 1, 1:200 in 5, 1:400 in 6, 1:800 in 10, 1:1,600 in 5, and 1:3,200 or higher in 4. None of these cats was ill, despite exhibiting high antibody titers. This is the first report of T. gondii infection in this host.

Coprology of Panthera tigris altaica and Felis bengalensis euptilurus from the Russian Far East.

Fecal samples from the Siberian tiger (Panthera tigris altaica) and the Amur cat (Felis bengalensis euptilurus) from Far Eastern Russia, were examined for parasites. A natural sedimentation methodology was used and a complete examination of all the sediment was performed. This fecal investigation allowed us to isolate and identify several developmental stages of gastrointestinal, hepatic, and respiratory parasites. Five parasites were found from P. t. altaica: 11 trematodes (Platynosomum fastosum) and 4 nematodes (Strongyloides sp., Ancylostomatidae, Toxascaris leonina, and Toxocara cati). Five parasites were found from F. b. euptilurus: 1 cestode (Diplopylidium sp.) and 4 nematodes (Trichuris sp., Ancylostomatidae, Toxascaris leonina, and Aelurostrongylus abstrusus). In addition, trophozoites of the amoeba Acanthamoeba sp. were detected in tiger feces.

Hepatozoonosis in two species of Japanese wild cat.

Hepatozoon sp. infections were detected in two species of Japanese wild cat, Iriomote wild cat (Felis iriomotensis) and Tsushima leopard cat (Felis bengalensis euptilura), between April 1993 and October 2005. The prevalence was 56.7% (17/30) and 14.3% (6/42), respectively. The most affected organ was the heart; all infected animals had organisms in their hearts. The parasitizing form was schizont and various developmental stages were observed. The size of schizont and merozoite was 22.3 +/- 3.1 x 15.3 +/- 2.2 mum and 6.1 +/- 0.6 x 2.3 +/- 0.2 mum, respectively. Few inflammatory reactions against the parasites were observed. Electron microscopically, organisms were located in parasitophorous vacuoles of unidentified host cells, and mature schizonts consisted of numerous merozoites. This is the first report of hepatozoonosis in Japanese felids.

Epidemiological survey of Trichinella infection in domestic, synanthropic and sylvatic animals from Argentina.

The presence of Trichinella larvae was investigated in 247 samples taken from domestic, synanthropic and sylvatic animals, collected during 1996 to 2005 in 12 endemic provinces of Trichinella infection in Argentina. Muscle larvae of Trichinella from 65 infected animals were identified at the species level by single larva nested polymerase chain reaction (PCR) technique based on the variability within the expansion segment V (ESV) region of the ribosomal DNA. Trichinella infections were found in 97 of 164 pigs, 38 of 56 pork products, two domestic dogs, one domestic cat, 7 of 11 armadillos and 3 of 9 synanthropic rats. All Trichinella isolates were identified as Trichinella spiralis by nested PCR. These findings add new data on the epidemiology of trichinellosis and should be considered when implementing new strategies to control this zoonosis.

Toxoplasmosis in Pallas' cats (Otocolobus manul) raised in captivity.

Manuls or Pallas' cats (Felis manul, syn. Otocolobus manul) are endangered wild cats from Central Asia kept and bred in many zoos. Despite good breeding success young cats frequently die from acute toxoplasmosis. From 1998 to 2002, a breeding pair in the Schönbrunn Zoo in Vienna, Austria, gave birth to 24 kittens; 58 % of kittens died between the 2nd and the 14th week of life, mostly due to acute toxoplasmosis. The epidemiology of toxoplasmosis in Pallas' cats was examined and a control strategy to protect the kittens from fatal toxoplasmosis was developed. One 12-week-old kitten from a litter of 6 born in 2001 died of generalized toxoplasmosis. This kitten had shed T. gondii oocysts that were bioassayed in mice. Toxoplasma gondii was isolated in tissue culture inoculated with tissues of these mice. The surviving animals were immediately treated with clindamycin for 16 weeks; they acquired a natural infection and seroconverted by the end of this time without clinical signs.